HK1161746B

HK1161746B - Multiplex amplification and detection

Info

Publication number: HK1161746B
Application number: HK12101720.1A
Authority: HK
Inventors: 富国良
Original assignee: 奥西泰克有限公司
Priority date: 2008-07-31
Filing date: 2009-07-30
Publication date: 2015-09-25

Abstract

The invention relates to the field of multiplex amplification. In particular, the invention relates to methods for assaying a sample for one or more nucleic acid targets in a single reaction based on the distinct melting temperatures or melting profiles of primers and/or probes. The invention also provides probes and kits for use in such methods.

Description

Multiplex amplification and detection

The present invention relates to the field of multiplex assays. The invention relates in particular to a method for detecting one or more target nucleic acids in a sample in a single reaction based on different melting temperatures or melting profiles of the probes. The invention also provides probes and kits for use in such methods.

Multiplex PCR, which utilizes multiple primer pairs to simultaneously amplify multiple target sequences in a single PCR reaction, is a more efficient PCR method than standard PCR, which uses a single primer pair. The simultaneous amplification of different target nucleic acids reduces the cost and time consumption of PCR analysis, minimizes the risk of experimental degradation and cross-contamination, and improves the reliability of the final result. Multiplex PCR has been used in a variety of fields of DNA detection, including identification of microorganisms, gene expression analysis, mutation and polymorphism analysis, genotyping and DNA array analysis, and RNA detection.

Real-time PCR has been developed for quantifying the amplification products during a PCR reaction. Real-time PCR is based on the principle that fluorescence emitted from a dye is directly or indirectly linked to the formation of a newly synthesized amplicon, or that annealing between a primer and a DNA template can be detected and is proportional to the amount of amplicon in each PCR cycle. Real-time PCR is performed in a closed tube manner and can be quantitative. Several methods are currently available for real-time PCR, such as using TaqMan probes (U.S. Pat. Nos. 5,210,015 and 5,487,972, and Lee et al, Nucleic Acids Res (Nucleic Acids research) 21: 3761-6, 1993), molecular beacons (U.S. Pat. Nos. 5,925,517 and 6,103,476, and Tyagi and Kramer, nat. Biotechnol. (national Biotech.) 14: 303-8, 1996), self-test amplicons (scorpion probes) (U.S. Pat. No. 6,326,145, and Whitcom et al, nat. Biotechnol. (national Biotech.) 17: 804-7, 1999), Amplisor (applied and environmental microbiology) 64: 4210-6, 1998), Amplifluor (U.S. Pat. No. 6,117,635, and Aczarnko et al, Res. Nucleic Acids research (Res. C.: Nucleic Acids research) 21: 5, Nucleic Acids replacement research (Nucleic Acids research) 5, Nucleic Acids research); DzyNA-PCR (Todd et al, Clin. chem. (clinical chemistry) 46: 625-30, 2000), fluorescence restriction enzyme detection (Cairns et al, biochem. Biophys. Res. Commun. (Biochemical and biophysical research communications) 318: 684-90, 2004), and proximity hybridization probes (U.S. Pat. No. 6,174,670 and Wittwer et al, Biotechniques 22: 130-1, 134-8, 1997). These probes mostly comprise a pair of dyes (one reporter dye and one acceptor dye) involved in Fluorescence Resonance Energy Transfer (FRET), wherein the acceptor dye quenches the fluorescent emission of the reporter dye. In summary, fluorescently labeled probes improve the specificity of amplicon quantification.

Another form of probe for use in PCR is a double-stranded linear probe having two complementary oligonucleotides. The probes described in the prior art are of the same length, with at least one oligonucleotide acting as a probe for a target sequence in a single stranded conformation. The 5 'end of one of the oligonucleotides is labeled with a fluorophore and the 3' end of the other oligonucleotide is labeled with a quencher, such as an acceptor fluorophore, or vice versa. When the two oligonucleotides anneal to each other, the two labels approach each other, thereby quenching fluorescence. Whereas a target nucleic acid that competitively binds to the probe will cause a less than proportional increase in fluorescence from the probe as its concentration increases. (Morrison L. et al, anal. biochem. (analytical biochemistry), Vol.183, pages 231. 244 (1989); U.S. Pat. No. 5,928,862).

Improved double-stranded linear probes are also known in the art, by shortening one of the complementary two oligonucleotides by several bases, thereby resulting in a partially double-stranded linearized probe. In the double-stranded linear probes of the prior art, the longer oligonucleotide end is labeled with a fluorophore and the shorter oligonucleotide end is labeled with a quencher. When the probe exists in a double-stranded form, the fluorescence emission is weak due to the close proximity of the fluorescent group and the quencher. And when a target nucleic acid is present, the shorter oligonucleotide with the quencher is replaced by the target nucleic acid. As a result, the fluorescence emission of longer oligonucleotides, which are present in the form of probe-target nucleic acid hybrids, is sufficiently enhanced (Li et al, nucleic acids Research, Vol.30, No.2, e5 (2002)).

US2005/0227257 describes a slightly improved double-stranded linear nucleic acid probe. The probe described in this patent application is improved by shortening one of the two complementary oligonucleotides by more bases than the aforementioned probe to obtain a partially double-stranded linearized probe.

Fluorescent hybridization probes are also used in other fields. For example, methods for multiplex genotyping using fluorescent hybridization probes have been described (e.g., US6,140,054) which use the melting temperature of a fluorescent hybridization probe that hybridizes to a target region of a genome, nucleic acid sequence, which is amplified by PCR, to identify mutations and polymorphisms.

The advent of high-throughput genetic testing necessitates the simultaneous qualitative and quantitative analysis of multiple genes and moves the combination of multiplex PCR and real-time PCR to multiplex real-time PCR. Due to the non-specificity of double-stranded DNA intercalating dyes, these dyes are not suitable for multiplex detection, whereas fluorescently labeled probes enable multiplex real-time PCR. However, multiplex real-time PCR is limited by the availability of fluorescent dye combinations. Currently, only four or five fluorescent dyes can be detected and quantified simultaneously at most in real-time PCR.

US2005/0053950 describes a method for multiplex real-time quantitative Polymerase Chain Reaction (PCR). The method relies on the different melting temperatures (T) of each amplicon when the amplicons are present in duplex or isolated form_m) And fluorescence emission changes of double-stranded DNA dyes, such as SYBR Green I, to quantify multiple PCR products or amplicons in a single real-time PCR reaction. For a specific amplicon at a temperature below its T_mMeasured at a temperature above T, and_mthe difference in fluorescence emission between the fluorescence emissions measured at the temperature of (a) corresponds to the value of the fluorescence emission of the amplicon present as a duplex. Accordingly, the difference in fluorescence emission of each amplicon in a single PCR reaction can be used to quantify it. However, the multiplicity and sensitivity of such methods is relatively low. For example, amplicons between about 100 to 150 nucleotides in length differ only slightly in melting temperature. Thus, these techniques require the use of amplicons of widely different sizes to be able to distinguish them.

However, for multiplex real-time PCR with higher multiplexing and sensitivity levels, there is also a need to develop other methods for amplifying and quantifying multiple target sequences in a single PCR reaction.

The method of the present invention is different from the prior art. First, the method is based on the fact that each probe has a different melting property (T)_mOr a melting map) And the fluorescence emission of the label on the probe changes when the double stranded portion inside the probe exists in duplex or isolated form. The probe of the present invention comprises a double-stranded portion which may be formed of a first oligonucleotide and a second oligonucleotide; for each probe, the double-stranded portion has a unique T_mThus, in a set of probes comprising the same or similar labels, different probes may be distinguished. Second, the first oligonucleotide may or may not comprise one or more labels, and this first oligonucleotide is consumed during the amplification reaction. The difference between the fluorescence emissions detected at two different temperatures corresponds to the fluorescence emission value of some of the probes when they are consumed. Third, melting profile measurements of unconsumed probe provide an indication of the presence or amount of the target nucleic acid in a sample.

To facilitate an understanding of the invention, some terms are defined below.

The term "nucleic acid" as used herein is a covalently linked sequence of nucleotides in which the 3 'position of the pentose of one nucleotide is linked to the 5' position of the next pentose by a phosphodiester group and in which the nucleotide residues (bases) are linked in a specific sequence, i.e. the linear order of the nucleotides. The term "polynucleotide" as used herein is a nucleic acid comprising a sequence greater than 200 nucleotides in length. The term "oligonucleotide" as used herein is a short polynucleotide or a portion of a polynucleotide. Oligonucleotides typically comprise a sequence of about 200 to about 100 bases. "nucleic acid", "DNA" and similar terms also include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, so-called "peptide nucleic acids" known in the art and having peptide bonds in the backbone rather than phosphodiester bonds are considered to be within the scope of the present invention.

As used herein, the terms "target sequence", "target nucleic acid sequence" and "nucleic acid of interest" are used interchangeably and refer to a segment of a target region to which amplification or detection, or both, is to be performed. The target sequence as the subject of amplification and detection may be any nucleic acid. The target sequence may be RNA, cDNA, genomic DNA, or DNA or RNA from, for example, a pathogenic microorganism or virus. The target sequence may also be DNA treated with chemical agents, various enzymes, and physical exposure. The target nucleic acid sequence of interest in a sample may be present as single stranded DNA or RNA such as cDNA, mRNA, other RNA, or as an isolated complementary strand. Separation of the complementary strand of the target nucleic acid can be achieved by physical, chemical or enzymatic means. For ease of description and understanding, reference to a nucleic acid of interest or a target nucleic acid refers to both those portions found in a test sample and to amplified copies of those portions of the nucleic acid, unless specifically stated to the contrary.

The term "primer" as used herein refers to a naturally occurring or synthetic oligonucleotide that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, i.e., in the presence of a nucleotide and a polymerization reagent, e.g., a DNA polymerase, and suitable temperature and buffer conditions. The primers herein are selected to be sufficiently complementary to the different strands of each specific sequence to be amplified. That is, the primers must be sufficiently complementary to their respective counterparts to hybridize therewith. A non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the diagnostic region of the target base sequence. The primers are generally complementary except when non-complementary nucleotides are present at the predetermined primer ends as described above.

The term "complementary to … …" as used herein means that one nucleotide can base pair with another specific nucleotide. That is adenosine is complementary to uridine or thymidine and guanosine is complementary to cytidine. For the purposes of this specification it is to be understood that thymidine and guanosine, although in some cases they may base pair, should not be considered complementary. For the purposes of the present invention, the term "sufficiently complementary" means that greater than or equal to 70%, preferably greater than 80%, more preferably greater than 90%, and most preferably greater than 95% or 99% of the nucleobases on one strand of a probe are able to find their Watson-Crick binding partners on the other strand of the probe (or the nucleic acid of interest) in a matched manner such that the corresponding nucleotides are able to hybridize to each other. The methods of determining the same or similar are encoded in publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, but are not limited to: the GCG Pileup program, which can be found in the GCG Pileup package, uses the Needleman and Wunsch algorithms with gap creation dependency 12 and gap extension dependency 4 as standard default values for the algorithms (Devereux et al, Nucleic Acids research Res 12: 387-395(1984)), BLASTP, BLASTN, and FASTA (Pearson et al, proc.natl.acard.sci.usa (proceedings of the american academy of sciences) 85: 2444-2448 (1988)). The BLASTX program is publicly available from NCBI and other sources (BLAST handbook, Altschul et al, Natl.Cent.Biotechnol.Inf (national center for Biotechnology information), Natl.Library Med. (national library of medicine) (NCBI NLM), NIH (national institute of health, USA), Bethesda, MD; Altschul et al, J.MoI.biol. (J. Mobiol.) 215: 403-410 (1990); Altschul et al, Nucleic Acids Res (Nucleic Acids research) 25: 3389-3402(1997)).

The terms "duplex" and "double-stranded" are used interchangeably and refer to the hybridization of one oligo-polynucleotide to a complementary oligo-polynucleotide.

The term "identical" means that two nucleic acid sequences have the same sequence or complementary sequences.

The term "homologous" means that a single-stranded nucleic acid sequence can hybridize to a complementary single-stranded nucleic acid sequence. The extent of hybridization can depend on a number of factors, including the amount of identity between the two sequences and the hybridization conditions (e.g., temperature and salt concentration). Preferably, the identical region is greater than about 5bp, more preferably, the identical region is greater than 10 bp.

As used herein, the term "continuous monitoring" and similar terms, refers to multiple monitoring during one cycle of PCR, preferably collecting at least one data point during a temperature transition, more preferably during each temperature transition.

The term "cycle-by-cycle" monitoring, as used herein, refers to one or more monitoring of a PCR reaction at each cycle.

The term "actual consumption" (ACA) refers to the amount of probe consumed as reflected by fluorescence detection in a reaction.

The term "amplification" as used herein refers to any amplification process that results in an increase in the concentration of a particular nucleic acid sequence in a mixture of nucleic acid sequences.

The term "sample" is used herein in its broadest sense. A biological sample suspected of containing nucleic acids may include, but is not limited to, genomic DNA, cDNA (in solution or bound to a solid support), and the like.

The term "label" as used herein refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) signal and that can be associated with a nucleic acid or protein. The signal provided by the label can be detected by fluorescence, radioactivity, colorimetry, gravimetry, magnetism, enzymatic activity, and the like.

The term "adjacent" or "substantially adjacent", as used herein, refers to the positional relationship of two oligonucleotides on the strand to which the template nucleic acid is complementary. The two regions of the template to which the oligonucleotide hybridizes may be contiguous, i.e., there is no space between the two template regions. Alternatively, the two regions of the template hybridized to the oligonucleotide may be separated by 1 to about 40 nucleotides, more preferably about 1 to 10 nucleotides.

The terms "thermal cycling", "thermal cycling" and "thermal cycling" refer to repeated cycles of temperature changes from a full denaturation temperature, to an annealing (or hybridization) temperature, to an extension temperature, and back to the full denaturation temperature. The above term also refers to repeated cycles from a denaturing temperature to an extension temperature, wherein the annealing and extension temperatures are combined to the same temperature. The complete denaturation temperature allows all double-stranded fragments to melt into single strands. The annealing temperature is such that the primer hybridizes to or anneals to a complementary sequence on an isolated single strand of a nucleic acid template. The extension temperature allows synthesis of the nascent DNA strand of the amplicon.

The term "reaction", as used herein, refers to a hybridization, extension or amplification reaction, or other biological, chemical reaction.

The term "amplification mixture" or "PCR mixture", as used herein, refers to a mixture of components required for the detection of a target nucleic acid from a nucleic acid template. The mixture may comprise nucleotides (dNTPs), probes, a thermostable polymerase, primers, and a plurality of nucleic acid templates. The mixture may further comprise a Tris buffer, a monovalent salt and Mg²⁺. The concentration of each component is well known in the art and can be further optimized by one of ordinary skill in the art.

The term "amplification product" or "amplicon" refers to a DNA fragment that is amplified by a polymerase in an amplification method, such as PCR, using a pair of primers.

The term "melting map" refers to a collection of measurements made on an oligo (or poly) nucleotide and its complement as an indication of the transition of the oligo (or poly) nucleotide molecule from a double stranded state to a single stranded nucleic acid (or vice versa). The transition of a nucleic acid from a double stranded state to a single stranded state is commonly described in the art as "melting" of the nucleic acid molecule. The above-mentioned transition can also be described as "denaturation" or "melting" of the nucleic acid. Accordingly, the melting profile in the present invention may also be referred to as "melting profile", "denaturation profile", "melting curve", "hybridization/melting profile", and the like.

The "melting temperature" or "T" of a nucleic acid molecule_m"generally refers to the temperature at which a polynucleotide dissociates from its complementary sequence. In general, T_mCan be defined as when a double-stranded nucleic acid moleculeThe temperature at which half of the Watson-Crick base pairs break or dissociate (i.e., "melt") while the other half of the Watson-Crick base pairs remain in a fully double-stranded conformation. In preferred embodiments the double stranded nucleic acid molecule is an oligonucleotide, in other embodiments the double stranded nucleic acid dissociates in a two state manner, in these embodiments, T of a nucleic acid_mIt can also be defined as the temperature at which half of the nucleic acid molecules in one sample are in a single-stranded conformation and the other half of the nucleic acid molecules in the sample are in a double-stranded conformation. Thus, T_mThe midpoint of the transition of the nucleic acid molecule from double stranded to single stranded (or vice versa) is indicated. It is generally accepted in the art that the transition of a nucleic acid molecule from double stranded to single stranded does not occur at a single temperature, but rather occurs over a range of temperatures. Nevertheless, T_mA convenient approximate measure of whether nucleic acid molecules in a sample are present in a single-stranded or double-stranded conformation is provided. Thus, the melting temperature of a nucleic acid sample can be readily obtained by analyzing the melting profile of the sample.

The term "consumption" or "consumption amount" refers to the reduction in the amount of free labeled probe at a temperature at which the labeled probe will generally remain intact. The labeled probe may not comprise a double-stranded portion; consumption of the labeled probe as described above may result in a change in the detection signal. Consumption of the labeled probe may include hybridization of the probe to the target nucleic acid or degradation of the probe upon hybridization to the target nucleic acid. When the probe comprises a double-stranded portion, the reduction in the amount of free labeled probe may be caused by the disappearance of at least one strand of the probe, i.e., the first oligonucleotide or the second oligonucleotide or both of the probes. The disappearance of at least one strand of the probe means that the first oligonucleotide of the probe or the second oligonucleotide of the probe or both oligonucleotides of the probe hybridize with the target nucleic acid. The hybridization of the first oligonucleotide of the probe or the second oligonucleotide of the probe or both oligonucleotides of the probe with the target nucleic acid may be followed by extension using the oligonucleotide of the probe as a primer or by degradation of the oligonucleotide of the probe.

The methods described herein allow for the substantially simultaneous amplification and detection of a large number of different target nucleic acid sequences.

In a first aspect, the present invention provides a method of detecting one or more target nucleic acids from a sample, the method comprising:

(a) contacting a sample comprising one or more target nucleic acids with a reaction mixture comprising:

a probe set comprising two or more probes, wherein at least one probe having a double-stranded portion may comprise

A first oligonucleotide comprising a first region and a second region, wherein said first region is sufficiently complementary to a portion of a target nucleic acid, and

at least one second oligonucleotide comprising a region sufficiently complementary to said second region of said first oligonucleotide,

such that said first and second oligonucleotides are capable of forming a double stranded portion of the probe,

wherein each probe comprises a detectable label or combination of detectable labels that produce a variable signal reflecting the presence or absence of a target nucleic acid, and

wherein at least two of the probes comprise the same detectable label or different detectable labels whose emission spectra are indistinguishable, and wherein each of the probes has a different melting property (melting temperature T)_m) And can be distinguished in the analysis of melting maps;

(b) performing a reaction in the sample/reaction mixture, wherein said reaction is a primer extension reaction under extension conditions, wherein, when a target nucleic acid is present, said probe is extended as a primer and thereby consumed, wherein a first oligonucleotide of a corresponding probe, which is an extendable primer, hybridizes to the target nucleic acid sequence and thereby is consumed in said primer extension reaction, wherein said consumed oligonucleotide of the probe is no longer able to participate in the formation of a double stranded portion (duplex) of the probe; and

(c) measuring the melting profile of the unconsumed probe in the reaction mixture at least once as a function of temperature by detecting a signal from the label on the unconsumed probe,

wherein the melt map provides an indication of whether at least one (target) nucleic acid of interest is present in the sample.

In this embodiment, the first oligonucleotide of the probe functions as a primer. In one primer extension reaction, a mixture of probes is added to a reaction mixture containing all the components for extension under extension conditions. If a particular target nucleic acid is present in the reaction, the oligonucleotide of the corresponding probe hybridizes to the target sequence and is then extended and incorporated into the primer extension product and is thereby consumed. The consumed oligonucleotide can no longer participate in the formation of the double stranded portion of the probe. In melt mapping analysis, the consumed probe appears as a decrease or disappearance of one peak.

In another aspect, the present invention provides a method of detecting one or more target nucleic acids from a sample, the method comprising:

(a) contacting a sample comprising one or more target nucleic acids with a hybridization reaction mixture comprising:

a set of probes comprising two or more probes, wherein at least one probe comprises

A first oligonucleotide comprising a first region substantially complementary to a portion of a target nucleic acid, and a second region, and

such that the first and second oligonucleotides are capable of forming a double stranded portion,

wherein said at least one probe comprises a detectable label or combination of detectable labels capable of producing a variable signal reflecting the presence or absence of a double stranded portion of the first and second oligonucleotides of the probe, and

wherein at least two of said probes comprise the same detectable label or different detectable labels whose emission spectra are indistinguishable, and wherein the double-stranded portion of the first and second oligonucleotides of each of said probes has a different melting characteristic and is distinguishable in a melt map analysis;

(b) performing a hybridization reaction in the sample/reaction mixture under hybridization conditions, wherein, when a target nucleic acid is present, a first oligonucleotide of the probe, which is sufficiently complementary to a portion of the target nucleic acid, hybridizes to the target sequence and is thus consumed during the above reaction, wherein said consumed oligonucleotide of the probe can no longer participate in the formation of the double-stranded portion (duplex) of the probe; and

In one embodiment, the probe or the first oligonucleotide of the probe may function as a hybridization probe. In one hybridization reaction, a mixture of probes is added to a reaction mixture containing all the components used under hybridization conditions. If a particular target nucleic acid is present in the above reaction, the oligonucleotide of the corresponding probe hybridizes to the target sequence and is thus consumed. The consumed oligonucleotide can no longer participate in the formation of the double stranded portion of the probe. In melt mapping analysis, the consumed probe appears as a decrease or disappearance of one peak.

(a) contacting a sample comprising one or more target nucleic acids with an amplification reaction mixture comprising:

(i) one or more pairs of forward/reverse oligonucleotide primers, wherein said primer pairs amplify one or more target nucleic acids when present in said sample,

(ii) a set of probes comprising two or more probes, wherein at least one probe comprises

wherein at least two of the probes comprise the same detectable label or different detectable labels whose emission spectra are indistinguishable, and

wherein the double stranded portion of the first and second oligonucleotides of each of said probes has a different melting characteristic and is distinguishable in a melt map analysis;

(b) performing an amplification reaction in a sample/amplification reaction mixture

Wherein, when a target nucleic acid is present, said first oligonucleotide, which is sufficiently complementary to a portion of the target nucleic acid, hybridizes to the target sequence and is thereby consumed during the amplification reaction;

(c) measuring the melting profile of the unconsumed probe at least once as a function of temperature by detecting a signal from a label on the unconsumed probe,

wherein the melting profile provides an indication of whether at least one target nucleic acid is amplified in the sample/amplification reaction mixture.

Wherein a first probe of the at least two probes has a melting temperature T based on its double-stranded portion_m1，

Wherein a second probe of the at least two probes has a melting temperature T based on the double-stranded portion thereof_m2，

Wherein T is_m1＞T_m2，

Wherein the same label is associated with said first and second probes respectively,

wherein, any is at T_m1 and/or T_m2 provides an indication of the consumption of the first and/or second probe.

Preferably, the method described above comprises step (d):

(i) comparing at least two melt profiles obtained in step (c)

And/or

(ii) Combining a melting profile obtained in step (c) with

A previously obtained melting profile of the same probe, or

A melting profile of the same probe obtained simultaneously in a parallel control reaction, or

A theoretical melting map of the same probe

Make a comparison

Wherein a change in said melting profile provides an indication of whether at least one target nucleic acid is amplified in said sample/amplification reaction mixture.

The amplification reaction may be any one of amplification methods such as PCR, SDA, NASBA, LAMP, 3SR, ICAN, TMA, helicase-dependent isothermal DNA amplification and the like. PCR is a preferred method of amplification.

The amplification reaction mixture should contain standard amplification reagents. Amplification reagents can be conveniently divided into four classes of components: (i) an aqueous buffer, typically including but not limited to a magnesium salt, (ii) an amplification substrate, such as DNA or RNA, (iii) one or more oligonucleotide primers (typically two primers for each target sequence, which when PCR is employed define the 5' ends of the two complementary strands of a double stranded target sequence), and (iv) an amplification enzyme such as a polynucleotide polymerase (e.g., Taq polymerase for PCR or RNA polymerase for TMA), or a ligase. In addition, appropriate nucleoside triphosphates are often required. The addition of further agents or additives may be determined by those skilled in the art as appropriate, and the selection of such agents is within the skill of those of ordinary skill in the art. Of course, when the amplification reagent is used for simultaneous reverse transcription and amplification, the amplification reagent further includes a reverse transcription reagent. The choice of amplification reagents, depending on the amplification reaction method employed, is within the skill of one of ordinary skill in the art.

In the methods described herein, the sample provided is a sample suspected of containing the target nucleic acid or nucleotide variation of interest. The target nucleic acid contained in the sample may be double-stranded genomic DNA or, if necessary, cDNA, which is then denatured using any suitable denaturation method known to those skilled in the art, including physical, chemical or enzymatic methods. One preferred physical method for strand separation involves heating the nucleic acid to its complete (> 99%) denaturation. Typical thermal denaturation conditions include temperatures ranging from about 80 ℃ to about 105 ℃ and times ranging from seconds to minutes. As an alternative to denaturation, the target nucleic acid may be present in the sample in a single stranded form, e.g., a single stranded RNA or DNA virus.

The denatured nucleic acid strand is then incubated with an oligonucleotide primer and a probe under hybridization conditions that allow the primer or probe to bind to the single nucleic acid strand. In some embodiments of the invention, the annealed primers and/or probes are extended by a polymerizing agent. The template-dependent extension reaction of the oligonucleotide primer is catalyzed by a polymerization agent in the presence of appropriate amounts of the four deoxyribonucleoside triphosphates (dATP, dGTP, dCTP and dTTP) or analogs thereof as previously described, in a reaction medium containing appropriate salts, metal cations and a pH buffering system. Suitable polymerization agents are enzymes known to catalyze primer and template dependent DNA synthesis. Reaction conditions for catalyzing DNA synthesis using the DNA polymerase are well known in the art. The probe is consumed during the amplification process.

An amplification primer can be a target-specific primer comprising a 3' start portion complementary to a target region of a target nucleic acid. The amplification primers can also be universal primers having a sequence that is the same or substantially the same as the 5' universal portion of the target-specific primers. The reaction may include a plurality of primers for amplification of a plurality of target sequences. The 5 'universal portion of the plurality of primers can have substantially the same sequence composition that is identical or substantially identical to the 3' start portion of the universal amplification primer. Preferably, the primers are DNA primers, in particular those suitable for PCR amplification.

For SNP genotyping or detecting variant nucleotides, the amplification primer may be an allele-specific primer wherein one terminal nucleotide of the primer is selected to be complementary to one of the suspected variant nucleotide or the corresponding normal nucleotide, whereby when the primer anneals to a diagnostic region comprising a particular nucleotide, the extension product of the primer is synthesized, and when the primer anneals to a diagnostic region not comprising the particular nucleotide of the target nucleic acid sequence, the extension product is not synthesized.

Amplification reaction mixtures include primer pairs consisting of forward and reverse primers, such that if a target nucleic acid is present in the sample, the primer pair will be capable of amplifying the target nucleic acid, preferably in an exponential manner.

In some embodiments, the reaction mixture will contain from 1 to 50, 1 to 25, 1 to 20, or 1 to 10 primer pairs. In other embodiments, the reaction mixture will contain 5 to 50, 5 to 25,5 to 20, or 5 to 10 primer pairs. As described above, the forward or reverse primer of a particular primer pair can be a common primer that is common to more than one primer pair.

The amplification reaction mixture comprises a set of two or more probes. The probe comprises:

a second oligonucleotide comprising a region substantially complementary to said second region of said first oligonucleotide,

such that the first and second oligonucleotides are capable of forming a double stranded portion of the probe.

The first oligonucleotide must be capable of binding to at least a portion of the target nucleic acid under suitable hybridization conditions. Preferably, each first oligonucleotide is specific for only a portion of one target nucleic acid. The first oligonucleotide should have a first region whose nucleotide sequence is complementary or sufficiently complementary to the nucleotide sequence of a portion of a target nucleic acid. The length of this first complementary region is preferably 6 to 100 nucleotides, more preferably 15 to 30 nucleotides.

The total length of the first oligonucleotide is preferably 15 to 150 nucleotides, more preferably 17 to 100 nucleotides, and most preferably 20 to 80 nucleotides.

In some embodiments where the reaction includes primer extension or amplification, the portion of the target nucleic acid that is complementary to the first oligonucleotide must belong to or overlap with the sequence that will be amplified by the forward and reverse primers. Alternatively, the first oligonucleotide may be one of the amplification primers, e.g., a forward or reverse primer. In some embodiments, the first and/or second oligonucleotide is not a forward or reverse primer.

The second oligonucleotide comprises a region that is substantially complementary to a second region of the first oligonucleotide. The length of this second region is preferably 4 to 100 nucleotides, more preferably 15 to 30 nucleotides. The second region of the first oligonucleotide may or may not overlap with the first region of the first oligonucleotide.

The total length of the second oligonucleotide is preferably 6 to 150 nucleotides, more preferably 10 to 100 nucleotides, and most preferably 12 to 80 nucleotides.

The first and second oligonucleotides may comprise 1 to 5 or 1 to 10 or more nucleotides at the 5 'or 3' end which are not complementary to the target nucleic acid or the first oligonucleotide, respectively.

Oligonucleotide probes may comprise nucleotides, nucleotide derivatives, nucleotide analogs, and/or non-nucleotide chemical moieties. Modifications to the probes that may facilitate probe binding include, but are not limited to, the incorporation of positively charged or neutral phosphodiester linkages into the probes to attenuate repulsion between the probes and the polyanionic backbone of the target nucleic acid (see Letsinger et al, 1988, j. amer. chem. soc. (journal of the american chemical society) 110: 4470); incorporating into the probe an alkylated or halogenated group such as 5-bromouridine to promote base stacking; incorporating ribonucleotides into the probe to cause the probe to: the target nucleic acid duplex forms an "a" type structure with enhanced base stacking; replacing some or all of the adenosine in the probe with 2, 6-diaminopurine (aminoadenosine); and incorporation of nucleotide derivatives such as LNA (locked nucleic acid), PNA (peptide nucleic acid) or the like.

Typically the 3' end of the probe is "blocked" to prevent incorporation of the probe into the primer extension product. However, in some preferred embodiments of the invention, some probes also function as primers, and thus the 3' end of the probe is unblocked. "blocking" can be achieved by using non-complementary bases or by adding a chemical moiety, such as a biotin or phosphate group, to the hydroxyl group of the last nucleotide. Depending on the moiety selected, the chemical moiety may also serve a dual role as a label for subsequent detection or capture of nucleic acids associated therewith. Blocking can also be achieved by removing the 3 '-hydroxyl group or using nucleotides lacking the 3' -hydroxyl group, such as dideoxynucleotides.

It will be understood that the term "probe" refers to a plurality of such probes, i.e.a molecule in the reaction mixture that does not contain only the probe.

In some embodiments of the invention, the first region of the first oligonucleotide does not overlap or does not sufficiently overlap with the second region of the first oligonucleotide.

In other embodiments of the invention, the first region of the first oligonucleotide substantially overlaps with the second region of the first oligonucleotide, or the second region is comprised in the first regionIn the domain. In these embodiments, the first oligonucleotide hybridizes to the target sequence to form the T of the duplex_mPreferably higher than the T of the duplex formed by hybridization of said first oligonucleotide to said second oligonucleotide_mSuch that if a target nucleic acid is present, the first oligonucleotide forms a more robust hybrid with the target nucleic acid and thus melts at a higher temperature than the first/second oligonucleotide duplex.

Preferably, the T of the duplex formed by the first oligonucleotide and the target nucleic acid_mT of a duplex formed by the first oligonucleotide and the second oligonucleotide_mAt least 2 degrees or at least 5 degrees higher.

In other embodiments, the first oligonucleotide may comprise a third region that is identical or substantially identical to the sequence of a primer used for amplification.

At least one probe of the invention is capable of forming a double stranded portion. Due to the double-stranded portion, the probe has a melting temperature T_mAnd a characteristic melting profile. In particular, a mixture of probes of the invention also has a characteristic melting profile.

Melting temperature (T)_m) Is influenced by several factors including, but not limited to, salt concentration, DNA concentration, presence of denaturants, nucleic acid sequence, GC content, and length. Typically, each double-stranded nucleic acid probe has a unique T_m. Below a given T_mAt least 50% of the nucleic acid duplexes remain in duplex form. In contrast, above a given T_mAt temperatures of (3), it is expected that more than 50% of the nucleic acid duplexes will melt into two oligonucleotide singlestrands.

T of any given DNA fragment_mCan be determined by methods well known in the art. For example, one fragment of DNA T is determined in the art_mIn one method, a constant temperature chamber in an ultraviolet spectrophotometer is used, and the measurement is carried out with the temperature slowlyIncreasing the absorbance at 268 nm. Absorbance is plotted against temperature to obtain a sigmoidal curve with two plateaus. (see, e.g., FIG. 1). The middle point of the absorbance between these two plateaus corresponds to the T of the fragment_m. Alternatively, the first negative derivative of absorbance is plotted against temperature to obtain a normal distribution curve. The peak of the normal curve corresponds to T of the fragment_m。

T of a probe can also be determined by the nearest neighbor method_mOr a plurality of T's of a mixture of probes_mAnd also T for a probe in the presence of a double stranded DNA dye or label on the probe in a single reaction_mOr a plurality of T's of a mixture of probes_mA detailed or precise measurement is carried out. For example, a reaction mixture comprising a probe and appropriate buffers is heated from a hybridization temperature to a complete denaturation temperature at a rate of 0.01 ℃ to 3 ℃ per second. At the same time, the mixture is illuminated with light of a wavelength absorbed by the dye (label) and the fluorescence emission of the dye (probe) is detected and recorded as a fluorescence emission reading. Plotting the first negative derivative of the fluorescence emission readings with respect to temperature against temperature yields several normal curves, each peak of which corresponds to the actual T for that probe_m. The curve is also referred to as a "melt profile" or a "hybridization/melting profile". T of probe_mOr the melt map can also be estimated by a computer program based on theories well known in the art.

For a multiplex assay, a probe set consisting of multiple probes for multiple target sequences is included in one reaction. In one embodiment, different probes in a set of probes may comprise the same label or labels whose emission spectra are indistinguishable. Each probe in such a probe set should have a different T_mThereby enabling the respective melting maps to be distinguished from each other. Not only individual probes have one melting pattern, but also a mixture of a plurality of probes in the probe set has a melting pattern characteristic of the probe set. The reaction is mixedThe article may comprise a single stranded probe having no characteristic melting temperature.

According to the present invention, it is possible to analyze a plurality of target nucleic acid sequences in a single tube by designing a probe set composed of probes hybridizing with different target sequences and probes having different melting temperatures based on the double-stranded portion inside thereof. If a target sequence is present, its corresponding probe is consumed. The sequence of the target can then be determined based on a comparison between the melting profiles of the probe sets before and after the reaction. Advantageously, different probes in a probe set may be associated with the same label, thereby allowing monitoring at a single emission wavelength. In one embodiment, each probe in the set of probes is associated with the same label, e.g., a fluorescent energy transfer pair or contact quenching pair, more particularly, a first label that is a fluorophore and a second label that is a quencher. Alternatively, a plurality of probe sets may be associated with different pairs of labels, so that the individual probe sets may be distinguished from each other based on different emission spectra.

According to the present invention, the method for analyzing a plurality of target nucleic acids may use a mixture of a plurality of probes associated with different labels having distinguishable emission spectra, or a mixture of a plurality of probes associated with labels having the same or overlapping emission spectra, but distinguishable based on the difference in melting temperature of the double-stranded portion therein.

When a probe having a double-stranded portion is used, the probe may comprise two strands: a first oligonucleotide and a second oligonucleotide, wherein said first oligonucleotide comprises a first region that is substantially complementary to a target nucleic acid (see figure 4). In one embodiment, the probe comprises one first oligonucleotide and at least one second oligonucleotide (see fig. 4A to J). The first oligonucleotide comprises a first region that is sufficiently complementary to a target nucleic acid and a second region, wherein the second region is sufficiently complementary to one or more second oligonucleotides such that the first and second oligonucleotides are capable of binding to form a double stranded portion. The first and second regions may be arranged in any order, such as 5 'to 3' or 3 'to 5' (see fig. 4A), or one region may be contained within the other region (see fig. 4B). Preferably, if the first oligonucleotide serves as a primer, the first and second regions are arranged in 3 'to 5' order (see FIG. 4A).

In one aspect, the first region of the first oligonucleotide does not overlap or does not sufficiently overlap with the second region of the first oligonucleotide (see fig. 4A). In other words, the first region is complementary to a target sequence and the second region is not complementary to the target sequence. When the second region is not complementary to the target sequence, the different probes may have the same or substantially the same second region sequence, and the different probes of the probe set may have the same second oligonucleotide between them. When the set of probes has the same second oligonucleotide, the second regions of the first oligonucleotide of different probes in the set may differ in length and/or nucleotide sequence such that each probe has a different T_mAnd a melt map.

In another aspect, the first region of the first oligonucleotide substantially overlaps with, or is contained within, a second region of the first oligonucleotide (see fig. 4B), wherein the first oligonucleotide hybridizes to a target sequence to form the T of a duplex_mA T higher than a duplex formed by hybridization of the first oligonucleotide to the second oligonucleotide_mSuch that if a target nucleic acid is present, it forms a more robust hybrid with the first oligonucleotide of the probe, such that the target nucleic acid/first oligonucleotide duplex melts at a higher temperature than the second oligonucleotide/first oligonucleotide duplex. In this aspect, the first region may be longer than the second region, or the second region may comprise mismatched nucleotides upon hybridization to the second oligonucleotide. Binding of the first oligonucleotide to the target nucleic acid prevents binding of the second oligonucleotide of the probe to the first oligonucleotide. Preference is given toT of a hybrid of said first oligonucleotide and said target sequence_mT of a hybrid of said first oligonucleotide and said second oligonucleotide_mAt least 2 degrees higher. More preferably, T of a hybrid of said first oligonucleotide and said target sequence_mT of a hybrid of said first oligonucleotide and said second oligonucleotide_mAt least 5 degrees higher.

In yet another aspect, the first oligonucleotide comprises a third region that is identical or substantially identical to a primer sequence (see FIGS. 4C and E). The third region may or may not be complementary to the target sequence. The plurality of probes in the set of probes may comprise the same third region sequence. The primer having the same sequence as the third region may serve as a universal amplification primer. When the target nucleic acid-specific probe (as a primer) becomes insufficient for amplification over several cycles, the above-mentioned universal primer can be used as a succeeding primer for continuing amplification in the subsequent cycles.

In some embodiments of the invention, the first and second oligonucleotides are linked by a linking moiety. Such a linking moiety may comprise a nucleotide, a nucleotide derivative, a nucleotide analogue or a non-nucleotide chemical bond, in other words, the first and second oligonucleotides may be a stretch of linked oligonucleotides (FIG. 4K). In this embodiment, the probe may be understood as comprising only one stretch of the first oligonucleotide (see FIG. 4K) comprising self-complementary regions capable of forming a stem-loop structure, wherein the self-complementary regions are sufficiently complementary to each other to form a double-stranded portion of the probe. Wherein the stem portion may be located at any part of the oligonucleotide and is 4 to 20 nucleotides in length. The 3' portion of the oligonucleotide is preferably complementary to the target sequence. It may have a blunt end, or a3 'overhang or a 5' overhang. Blunt ends or 3' overhanging ends are preferred forms.

The first oligonucleotide of the above-mentioned probe containing a double-stranded portion can be consumed during the amplification. Alternatively, both the first and second oligonucleotides of the double-stranded portion-containing probe described above can be consumed during amplification. Preferably, the first oligonucleotide is designed to be consumed in one reaction, while the second oligonucleotide may remain unchanged.

The probe may be extended so as to function as a primer. Alternatively, the 3 'end of the first oligonucleotide is blocked and the 3' end of the second oligonucleotide is blocked and thus not extendable.

Each probe comprises a detectable label that produces a variable signal that reflects the presence or absence of the double-stranded portion of the probe.

Furthermore, at least two of the probes comprise the same detectable label or different detectable labels whose emission spectra are indistinguishable.

The label on the probe may be a fluorophore, or the probe may comprise a pair of interacting labels, such as a fluorophore and/or a non-fluorescent dye. An example of such an interactive label is a pair of fluorophore-quencher pairs. The label on the probe may be located at any position as long as it is capable of interacting with other labels or other entities such as G nucleotides.

In some embodiments, the first oligonucleotide comprises a first label and the second oligonucleotide comprises a second label. Preferably, the first label is a fluorophore and the second label is a quencher, or vice versa.

In other embodiments, the probe comprises two labels, the two labels being a FRET pair. Preferably, one label is on the first oligonucleotide and the second label is on the second oligonucleotide.

Furthermore, both the first and second oligonucleotides may comprise a plurality of tagging entities. For example, both the first and second oligonucleotides can comprise both a fluorescent group and a quencher.

Typically, the fluorescent gene and the quencher are associated with the oligonucleotide in a manner such that when the first oligonucleotide is bound to an unlabeled template sequence (e.g., a target nucleic acid), the fluorescent group is separated from the quencher.

Alternatively, the fluorescent gene and the quencher are associated with the oligonucleotide in a manner such that when the first oligonucleotide is bound to an unlabeled template sequence (e.g., a target nucleic acid), the fluorophore and the quencher are drawn into close proximity, thereby quenching the fluorophore.

As used herein, the term "fluorophore" refers to a group that absorbs light energy over a range of excitation wavelengths and emits light energy over a different range of wavelengths.

Examples of fluorescent labels include, but are not limited to: alexa Fluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPY FL, BODIPY 6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), carboxyrhodamine 6G, carboxy-X-Bluedamine (ROX), Cascade Blue (Cascade), Cascade Yellow (Yellow), cyanine dyes (Cy3, 5, Cy3.5, Cy5.5, Cy sulfonyl chloride, coumarin (2-D ', dimethoxy coumarin) 2 ' -fluorescein, fluorescent red-coumarin (2-D ', fluorescent red-coumarin), fluorescein, FAM, hydroxycoumarin, IRDye (IRD 40, IRD 700, IRD 800), JOE, lissamine rhodamine B, Marina Blue, methoxycoumarin, naphthalocoumarin (Naphthofluorescein), oregon Green 488, oregon Green 500, oregon Green 514, Pacific Blue (Pacific Blue), PyMPO, pyrene, rhodamine 6G, rhodamine Green, rhodamine red, p-methylaminophenol Green (Rhodol Green)2 ', 4', 5 ', 7' -tetrabromo sulfone-fluorescein (2 ', 4', 5 ', 7' -Tetra-bromosylfone-fluorescein), tetramethyl rhodamine (TMR), carboxytetramethyl rhodamine (TAMRA), texas red and texas red-X.

As used herein, the term "quencher" includes any group that, when in close proximity to a fluorescent label in an excited state, is capable of absorbing the energy of the fluorescent label and dissipating this energy. The quencher can be a fluorescent quencher or a non-fluorescent quencher, the latter also known as a dark quencher. The fluorophores listed above can function as quenchers when brought into proximity with another fluorophore, wherein the quenching that occurs can be FRET quenching or contact quenching. It is preferred to use a dark quencher which does not emit any visible light. Examples of dark quenchers include, but are not limited to: DABCYL (4- (4 ' -dimethyl p-aminoazobenzene) benzoic acid) succinimidyl ester, diarylrhodamine carboxylic acid (diarylrhodamine carboxylic acid), succinimidyl ester (QSY-7), 4 ', 5 ' -dinitrofluorescein carboxylic acid, succinimidyl ester (QSY-33), sequenchel, "black hole quenchers" (BHQ-1, BHQ-2 and BHQ-3), nucleotide analogs, guanylic acid residues, nanoparticles, and gold particles.

The interactive label pairs may form a FRET relationship or a contact quenching relationship. Preferably, for efficient contact quenching, the distance between the fluorescent label and the quencher is from 0 to 10 nucleotides; more preferably, the distance between the fluorescent label and the quencher is from 0 to 5 nucleotides; more preferably, the distance between the fluorescent label and the quencher is from 0 to 2 nucleotides. The quencher may be a nanoparticle. The nanoparticle may be a gold nanoparticle. The quencher may also be a guanylic acid residue or a polyguanylic acid residue.

The label or combination of labels on each probe is capable of generating a detectable signal characteristic of each probe, which signal is indicative of the presence or absence of the duplex region of the first and second oligonucleotides. The function of the label is to indicate whether the first oligonucleotide of the probe is bound to the second oligonucleotide or the target nucleotide, and further to indicate whether the second oligonucleotide is bound.

At least one label is associated with the probe or a first oligonucleotide thereof, or with a second oligonucleotide of the probe. The label increases or decreases the fluorescence emission when the first oligonucleotide binds to the second oligonucleotide.

Preferably, the probe comprises a first label and a second label, wherein at least one label is capable of producing a detectable signal, and wherein the intensity of the signal is affected by the proximity of the two labels.

In some embodiments of the invention, the first label is associated with one strand of the double-stranded portion and the second label is associated with the other strand of the double-stranded portion of the probe, such that the first and second labels are located in close proximity when an internal duplex of the probe is formed.

In other embodiments, the first label is associated with the first oligonucleotide and the second label is associated with the second oligonucleotide such that the first label and second label are located in close proximity when the internal double stranded portion of the probe is formed.

In some embodiments of the invention, the first oligonucleotide does not comprise a label. In other embodiments, the second oligonucleotide comprises a single label that alters its fluorescent emission when the second oligonucleotide is hybridized to the first oligonucleotide.

In some embodiments, the first label is associated with the first oligonucleotide and the second label is associated with the second oligonucleotide such that the first label and second label are located in close proximity when the internal duplex of the probe is formed. Preferably, the first label is associated with a second region of the first oligonucleotide and the second label is associated with a region of the second oligonucleotide complementary to the second region of the first oligonucleotide, such that the first and second labels are drawn together into close proximity when the internal duplex of the probe is formed. Examples of such embodiments are shown in fig. 4A, 4B and 4C.

In some aspects of the invention, the first oligonucleotide of the probe does not comprise a label, while the second oligonucleotide of the probe comprises at least one, preferably two labels.

In one embodiment of this aspect, the second oligonucleotide comprises a first label and a second label. Said first label being associated with said second oligonucleotide at or near one end thereof and said second label being associated with said second oligonucleotide at or near the other end thereof, whereby when the second oligonucleotide is not hybridised to the first oligonucleotide, the second oligonucleotide is in a random coil configuration or a stem-loop configuration which draws the first and second labels together in close proximity. The two labels remain remote from each other when the second oligonucleotide is hybridized to the first oligonucleotide. Examples of such embodiments are shown in fig. 4D, 4E and 4F.

It is known in the prior art that a ditag oligonucleotide can form a random coil structure when it is in a single stranded state and at a certain permissive temperature. Such linear oligonucleotide probes behave like a random coil in solution: its two ends may accidentally approach each other, resulting in a measurable change in energy transfer. However, when such a probe binds to its template, the probe-template hybrid forces the two ends of the probe apart, disrupting the interaction between the two end portions, resulting in a change in fluorescence emission.

Double-labeled oligonucleotides may also form a stem-loop structure known as a molecular beacon. Molecular beacon probes are single-stranded oligonucleotide probes that form a hairpin structure in which a fluorophore and a quencher are typically disposed at opposite ends of the oligonucleotide. The short complementary sequence at the probe end allows the formation of an internal stem of one molecule, allowing the fluorophore and the quencher to be in close proximity. The loop portion of the molecular beacon is complementary to a target nucleic acid of interest. The binding between this probe and its target nucleic acid forms a hybrid, allowing the stem to separate. This will result in a conformational change which separates the fluorophore and the quencher from each other and results in a stronger fluorescence signal (Tyagi S. and Kramer F.R., Nature Biotechnology (Nature Biotechnology), Vol.14, pages 303-308 (1996); Tyagi et al, Nature Biotechnology (Nature Biotechnology), Vol.16, pages49-53 (1998); Piatek et al, Nature Biotechnology (Nature Biotechnology), Vol.16, pages 359-363 (1998); Marras S. et al, Genetic Analysis: Biomolecular Engineering, Vol.14, pages 151 pp 156 (1999); Tras I. et al, Biotechnologies (Biotechnology), Vol 28, pages 738-2000) (732).

In the present invention, a second oligonucleotide of a linear probe having a double-stranded portion is a portion of a double-stranded portion of a probe, wherein the second oligonucleotide may be an oligonucleotide similar to a molecular beacon. Such probes differ from other probes in that the second oligonucleotide may not hybridize to the target sequence but to a second region of the first oligonucleotide, wherein the second region may be unrelated to the target sequence. The second oligonucleotide may be capable of hybridizing to the target sequence, but it is designed not to hybridize to the target sequence in the actual amplification reaction. The second oligonucleotide may comprise a sequence that does not bind strongly to a target sequence. During the extension and annealing steps of the amplification reaction, the temperature may be too high for the second oligonucleotide to hybridize to the target sequence. In the signal collection step, which is usually performed after the extension step, the temperature may be low, but since the target sequence to be amplified may become double-stranded due to the extension of the amplification primer, the target sequence may not be hybridized with the second oligonucleotide. Thus, at the signal collection step, the second oligonucleotide may only be able to hybridize to the first oligonucleotide where the probe is not consumed.

In another aspect, the first oligonucleotide is free of a label and the second oligonucleotide comprises a label. When the second oligonucleotide is hybridized to the first oligonucleotide to form the double-stranded portion of the probe, the detectable signal emission of the label is altered relative to the emission of the label in single-stranded form of the second oligonucleotide. This may be due to the label being pulled to a position immediately adjacent to one or more nucleotides in the first oligonucleotide, or due to the label being pulled away from one or more nucleotides in the second oligonucleotide.

It is known in the art that when a fluorescent dye is placed in close proximity to a particular nucleotide, such as a G nucleotide, its fluorescence emission can change.

In another embodiment, the first oligonucleotide of the probe is free of a label and the probe comprises two second oligonucleotides capable of hybridizing adjacent or sufficiently adjacent to different portions on the second region of the first oligonucleotide, wherein one of the second oligonucleotides is associated with a first label and the other second oligonucleotide is associated with a second label, such that when the two second oligonucleotides hybridize to the first oligonucleotide, the two labels are drawn into close proximity and one label affects the signal from the other label.

The close proximity may result in a relationship such as FRET or contact quenching. The two second oligonucleotides hybridized to the first oligonucleotides are part of the probe. The two labeled second oligonucleotides are designed not to hybridize to the amplified target sequence but to the first oligonucleotides that are not consumed. Examples of such embodiments are given in fig. 4I and 4J.

In other embodiments, the first and second oligonucleotides of a probe are linked by a linking moiety comprising a chemical moiety that is either a nucleotide or a non-nucleotide, thereby allowing the first and second oligonucleotides to form a stem-loop structure, wherein the first and second oligonucleotides are labeled separately, such that when the probe forms an internal stem-loop structure, the labels are pulled into close proximity and one label affects the signal from the other label.

The linking moiety may be of the formula (CH)₂)_nOr a functionally equivalent one (n is preferably 1 to 100 or 1 to 50). Preferably, the linker is an oligonucleotide to which the first and second oligonucleotides are linked. Preferably, the loop is complementary to the target sequence.

At least two probes in the amplification reaction mixture comprise the same detectable label or a different detectable label whose emission spectra are indistinguishable.

In a multiplex reaction, two or more probes are used to detect the presence of two or more target nucleic acids. However, this does not necessarily mean that each different probe needs to have a different distinguishable label. Each probe may have a unique melting profile that depends on the characteristics of its internal double stranded portion. Thus, if two or more probes have distinguishable melting characteristics, the same label may be used for the probes. That is, different probes labeled with the same label or a label whose emission spectra are indistinguishable must have different melting characteristics, preferably different melting temperatures (T)_m). The different melting characteristics described above will allow the various probes to be identified and/or quantified in step (c).

The term "melting property" as used herein includes the melting profile of the probe (preferably measured by measuring the signal from a label on the probe as a function of temperature) and/or the melting temperature (T) of the probe_m)。

When referring to the melting characteristics of the probes as being "different", it is understood that such differences are measured under the same or control conditions.

In some embodiments of the invention, two or more probes are labeled with the same detectable label. For example, at least 2, 3,4, 5, 6, 7, 8, 9, 10, 20, or 30 or more of the different probes of the first oligonucleotide can all be labeled with the same label, or at least 2, 3,4, 5, 6, 7, 8, 9, 10, 20, or 30 or more of the different probes of the second oligonucleotide can all be labeled with the same label.

In step (b), an amplification reaction is performed in the sample/amplification (or sample/hybridization) reaction mixture, wherein, when a target nucleic acid is present, a probe (first oligonucleotide) that is sufficiently complementary to a portion of the target nucleic acid is consumed during the amplification reaction.

The amplification reaction may be performed under conditions known in the art such that a probe sufficiently complementary to a portion of the target nucleic acid is consumed.

Preferably, the amplification comprises at least one denaturation step, at least one annealing step and at least one primer extension step.

More preferably, the amplification is a thermocycling amplification comprising two or more denaturation, annealing and primer extension steps.

Preferred amplification reactions include PCR, SDA, NASBA, LAMP, 3SR, ICAN, TMA, helicase dependent isothermal DNA amplification and the like. PCR is a preferred method of amplification. If the amplification is PCR, the conditions comprise subjecting the reaction to thermal cycling.

When a target nucleic acid is present in the sample, at least some of the probes (first oligonucleotides) complementary to portions of the target nucleic acid will hybridize to the respective complementary portions under appropriate reaction conditions. The first oligonucleotide will thus be consumed.

Consumption of the probe is achieved by its hybridization to the target sequence, which may be followed by incorporation of the probe into the amplification product or/and degradation of the probe during the amplification step. That is, the first oligonucleotide of the probe forms a portion of the probe before it can no longer be reconstituted after it has been consumed.

In an amplification reaction, when the probe has two strands, the probe of the present invention may be constituted by adding the first and second oligonucleotides to the reaction in an arbitrary ratio, for example, the ratio of the second oligonucleotide to the first oligonucleotide is preferably more than 1, or may be more than 0.1 and less than 1. Thus, the first and second oligonucleotides may be added independently to the amplification reaction mixture.

Depending on the type of detection method and the type of label actually used, the signal (e.g., fluorescence emission) from the probe may be increased or decreased when the probe or the first oligonucleotide of the probe is consumed. See the embodiment shown in figure 4. In fig. 4A to 4C, the consumption of the probe leads to an increase in fluorescence, since the first oligonucleotide associated with the fluorophore is consumed and thus the fluorophore is allowed to emit its signal. Conversely, in fig. 4D to 4F, consumption of the first oligonucleotide releases the dual end-labeled second oligonucleotide and thus allows the fluorophore and the quencher to be juxtaposed to each other, resulting in a decrease in the signal from the fluorophore.

In one embodiment, the probe first oligonucleotide may be extended and function as a forward or reverse primer. The first oligonucleotide of the probe may serve as one of the amplification primers, and may be incorporated into the amplification product and thereby consumed (see FIG. 6). In one PCR, the first oligonucleotide of the probe is amplified together with another amplification primer for the reverse strand as a primer pair. In the signal collection step, or the step of measuring the melting profile of the probe, the first oligonucleotide of the probe that has been incorporated into the amplification product is no longer able to participate in the formation of a duplex of the internal double stranded portion of the probe. The amount of uneaten first oligonucleotide of the probe that is capable of participating in the formation of the probe duplex can be determined, and this signal can be converted into the amount of consumed first oligonucleotide, thereby determining which or how much target sequence is present in a sample. When the first oligonucleotide functions as a primer, the second oligonucleotide preferably does not function as a primer.

In another embodiment of the invention, the single-stranded probe or the first oligonucleotide of the probe having two strands is degraded during the amplification process. For example, the first oligonucleotide may include a chemical moiety that is a nucleotide or a non-nucleotide that is sensitive to a digesting agent. In a further example, the probe or the first oligonucleotide is capable of being degraded by a digestion agent as described above when hybridized to a target sequence. For example, the probe or the first oligonucleotide may comprise an RNA nucleotide. When the first oligonucleotide is hybridized with a target sequence, it can be degraded by an enzyme having nucleic acid nuclease H activity. It may be designed such that the first oligonucleotide cannot be degraded when it is hybridized with the second oligonucleotide to form the double-stranded portion of the probe. The first oligonucleotide may also be degraded by an exonuclease. For example, the 3 'nucleotide of the first oligonucleotide may be degraded by the 3' exonuclease activity of a polymerase. The first oligonucleotide may also be degraded by an endonuclease, e.g., by a restriction enzyme upon hybridization to a target nucleic acid.

Preferably, the single stranded probe or the first oligonucleotide of the probe having two strands is degraded by the 5' exonuclease activity of a DNA polymerase, such as Taq DNA polymerase. In this embodiment, the first oligonucleotide of the probe may be blocked at the 3' end and therefore not extended. In PCR amplification, the first oligonucleotide hybridizes to regions of the target sequence flanked by the forward and reverse primers, respectively, and is degraded by a nuclease activity, such as the 5' exonuclease activity of Taq polymerase (see FIG. 8). Alternatively, the 3' end of the first oligonucleotide of the probe is unblocked and thus capable of being extended. The first oligonucleotide hybridizes to a target sequence and is capable of being extended by a polymerase. An amplification primer upstream of the first oligonucleotide is also extended. In PCR amplification, when the amplification primer extends to meet the extended strand of the first oligonucleotide, the entire extended strand of the first oligonucleotide is degraded by a nuclease activity, such as the 5' exonuclease activity of Taq polymerase (see FIG. 7).

In another embodiment of the invention, the consumption of the probe may simply be the hybridization of the probe to the target sequence, whereby the hybridized probe is no longer able to form the double stranded structure of the probe (see FIG. 5). Preferably, the amplification is designed to produce a single stranded product such that the first oligonucleotide of the probe is capable of hybridizing to the target sequence during the annealing step and/or after the extension step. The method used to produce single stranded products may be asymmetric PCR, or one of the methods described in PCDR (PCT/GB 2007/003793). The single-stranded amplified strand may form a strong hybrid with the first oligonucleotide; the first oligonucleotide can therefore be considered to have been consumed because it is difficult to re-supply the internal hybrid forming the probe.

In another embodiment of the invention, the first oligonucleotide of the probe may function as a nested inboard amplification primer. The first oligonucleotide anneals to the same strand of the target nucleic acid as one of the outer amplification primers. After hybridization to the target nucleic acid, both the outer amplification primer and the first oligonucleotide may be extended. If the DNA polymerase comprises a displacement activity, the extended strand of the first oligonucleotide may be displaced during extension of the outer primer. If the DNA polymerase comprises a 5' exonuclease activity, the extended strand of the first oligonucleotide may be degraded during extension of the outer primer.

In some embodiments, the amplification conditions may be designed such that the probe is not consumed at some stages of the amplification, even when the target nucleic acid is present, and is consumed at other stages. For example, if the amplification is PCR, the probe may be set too high for the annealing and extension temperatures to bind and not be consumed during some thermal cycles of amplification. Thermocycling conditions for amplification can be designed such that the probe can be consumed at some stage or the last cycle of amplification. For example, after thermal cycling, the PCR tube is incubated at a temperature lower than both the annealing and extension temperatures of the thermal cycling. This lower temperature allows the probe to hybridize to the target nucleic acid, resulting in extension or degradation of the hybridized probe.

Step (b) may further comprise step (b 1): fluorescence Emissions (FEs) are measured cycle by cycle at a plurality of Measurement Temperatures (MTs). The Measured Temperatures (MTs) are the temperatures at which the label on the probe is read for fluorescence emission on a cycle-by-cycle basis to determine the amount of fluorescence emission from one probe.

Preferably, the fluorescence emission of a label on said probe is obtained, detected and/or recorded after the reaction mixture has been irradiated or excited with light of a wavelength absorbed by the label at each cycle of a reaction. The term "cycle by cycle" refers to measurements in each cycle. In one cycle, fluorescence emission readings are taken at one measurement temperature to calculate the amount of fluorescence emission from the remaining probes. The fluorescent emission may be detected, recorded or obtained continuously or intermittently.

During a continuous recording process, the fluorescence emission of the probe is monitored and recorded, for example, every 50ms, every 100ms, every 200ms or every 1s, at each cycle of, for example, a PCR reaction. A perspective view of time, temperature and fluorescence emission can thus be formed. At any given cycle, a fluorescence emission reading is taken at a time point corresponding to a desired MT to determine the amount of fluorescence emission from the probe in that cycle. During an intermittent recording, fluorescence emission readings are taken only when the reaction temperature reaches a desired MT during each cycle.

For probes that are consumed by incorporation into amplification products or degradation by a digestion agent, the cycle-by-cycle Fluorescence Emission (FE) is preferably measured after the completion of the extension step of each cycle. For probes that are consumed by hybridization to a target sequence, the cycle-by-cycle Fluorescence Emission (FE) can be measured before the extension step of each cycle is completed.

Fluorescence Emission (FE) as used herein refers to baseline corrected fluorescence (dR). Typically, for each well (reaction) and each light path, a linear least mean square algorithm (or other similar algorithm) is used to fit the raw fluorescence data over a range of cycles to obtain a baseline. For each cycle, the value of the baseline function is calculated and subtracted from the original fluorescence to obtain baseline-corrected fluorescence (dR).

The fluorescence intensity data (amplification plot) can be described as a binary function: a linear component or background, and an exponential component containing pertinent information. The linear portion of the fluorescence can be calculated and subtracted to isolate the above-mentioned exponential component. For each amplification plot (i.e., each reaction and each label), the treatment performed consisted of three steps:

1. a range of cycles was identified where all fluorescent components were strictly linear (no exponential increase in fluorescence).

2. Using the fluorescence intensity values determined above over the course of the cycle, the data were fitted to a straight line (a function that predicts the contribution of the linear component over the entire reaction).

3. During each cycle, the predicted background fluorescence intensity was subtracted. The curve thus obtained corresponds to the change in fluorescence due to DNA amplification.

When the amplification reaction mixture contains "n" kinds of probes for multiplex detection of "n" kinds of target nucleic acids, the melting temperature of the first probe is T_m1, the second probe has a melting temperature T_m2, the melting temperature of the third probe is T_m3, the melting temperature of the kth probe is T_mk and the melting temperature of the nth probe is T_mn, wherein T_m1＞T_m2＞T_m3＞…T_mk…＞T_mn, wherein n and k are positiveAn integer, k is greater than or equal to 1 and less than or equal to n, and n is greater than or equal to 2.

The percentage of the double-stranded form of a probe to the total amount of probe at a particular temperature or set of temperatures can be determined experimentally or predicted, where the prediction can be made by a computer program. Since a first negative derivative of fluorescence emission when a probe melts versus temperature yields a normal distribution curve, one of ordinary skill in the art of statistics can readily define an MT at which a percentage of a given total amount of probe is in either double-stranded or single-stranded (i.e., isolated) form. Accordingly, a measured temperature is one at which, for example, no more than 20% of a probe is in single stranded form. A table can be prepared listing the percentage of double-stranded (ds) and single-stranded(s) forms for each probe at each temperature.

A first fluorescence emission FEa can be obtained at a measurement temperature MTa, where more than 50% of the first probe at MTa is in double-stranded form; a second fluorescence emission FEb can be obtained at a measurement temperature MTb, where more than 50% of the second probe is in double-stranded form at this MTb; (ii) a kth fluorescence emission FEk can be obtained at a measurement temperature MTk, wherein more than 50% of the kth probe is in double-stranded form at this MTk; (ii) an n-1 fluorescence emission FE (n-1) can be obtained at a measurement temperature MT (n-1), wherein more than 50% of the n-1 probes of MT (n-1) are in double-stranded form; obtaining an nth fluorescence emission FEn at a measurement temperature MTn, wherein more than 50% of the nth probe is in double-stranded form at the MTn; alternatively, a fluorescence emission FE0 can be obtained at a measurement temperature MT0, wherein no more than 10% of the first probe is in double-stranded form at this MT 0.

Although 50% is mentioned above as a preferred amount of probe in double stranded form, it is contemplated that any percentage may be used, for example 40%, 55%, 70% or 80%. Preferably, the step of obtaining the fluorescence emission FEk cycle by cycle at a measurement temperature MTk at which no more than 30% of the (k-1) th probe is in its internal double-stranded form. More preferably, the step of obtaining the fluorescence emission FEk cycle by cycle at a measurement temperature MTk at which no more than 20% of the (k-1) th probe is in its internal double-stranded form at the MTk.

Step (b) may further comprise step (b 2): for each probe, the actual consumption of fluorescence emission from the consumed probe was determined cycle by cycle, where the actual consumption of fluorescence emission of the kth probe was recorded as ACA_k. At a specific measurement temperature (MTa), at which a certain percentage (dska)% of one probe is in ds (double stranded) form, the first probe contributes (ds1 a)% (ACA) to the fluorescence emission FE at the measurement temperature MTa₁) The contribution of the second probe was (ds2 a)% (ACA)₂) The contribution of the kth probe was (dska)% (ACA)_k). For example, at 60 ℃, 70% of probe 1 is in the ds form; at 50 ℃, 80% is in the ds form. The contribution of probe 1 to FE at 60 ℃ was 70% (ACA)₁) (ii) a The contribution of probe 1 to FE at 50 ℃ was 80% (ACA)₁). If there are multiple probes, FE is the total amount contributed by all probes consumed. The Actual Consumption (ACA) can be calculated by the following formula:

at a temperature a, the total fluorescence emission is

FEa＝(ACA1)*(ds1a)％+(ACA2)*(ds2a)％+(ACA3)*(ds3a)％…+(ACAn)*(dsna)％

At temperature b, the total fluorescence emission is

FEb＝(ACA1)*(ds1b)％+(ACA2)*(ds2b)％+(ACA3)*(ds3b)％…+(ACAn)*(dsna)％

At a temperature c, the total fluorescence emission is

FEc＝(ACA1)*(ds1c)％+(ACA2)*(ds2c)％+(ACA3)*(ds3c)％…+(ACAn)*(dsna)％

And so on. The ACA alone can be calculated from the above formula.

"" indicates "multiplied".

Preferably, the actual consumption of each probe is obtained by means of a computer program running the above calculations. Alternatively, the above calculation may be performed manually.

For example, in one amplification reaction, there are three probes for three target sequences. At 65 ℃, 5% of the first probe is in double-stranded form, 0% of the second probe and 0% of the third probe are in double-stranded form. At 60 ℃, 60% of the first probe is in double-stranded form, 5% of the second probe is in double-stranded form, and 0% of the third probe is in double-stranded form. At 55 ℃, 90% of the first probe is in double-stranded form, 55% of the second probe is in double-stranded form, and 5% of the third probe is in double-stranded form. At 45 ℃, more than 95% of all the various probes are in double-stranded form. The first fluorescence emission FE60 was collected at 60 ℃, the second fluorescence emission FE55 was collected at 55 ℃ and the third fluorescence emission FE45 was collected at 45 ℃. Alternatively, one fluorescence emission collected at a temperature of 65 ℃ or higher prior to collecting the first fluorescence emission is FE 65. The FE contributed by the actual ACA consumption of the individual probes was calculated as ds% > (ACA). See table below:

FE65＝5％*ACA1+0％*ACA2+0％*ACA3≈0

FE60＝60％*ACA1+5％*ACA2+0％*ACA3 (1)

FE55＝90％*ACA1+55％*ACA2+5％*ACA3 (2)

FE45＝95％*ACA1+95％*ACA2+95％*ACA3 (3)

the ACA alone can be calculated from the above formula. If we assume 5% ACA is negligible. The approximate ACA can be calculated from (1), (2) and (3), wherein

ACA1＝(EF60)/0.6

ACA2＝((EF55)-0.9/0.6*(EF60))/0.55

ACA3＝(EF45)/0.95-(EF60)/0.6-(EF55-(0.9/0.6)*(EF60))/0.55

As thermal cycling of the PCR mixture proceeds, the fluorescence emission and Actual Consumption (ACA) are recorded (calculated) and plotted against cycle number to obtain a plot of fluorescence emission versus cycle. The amount of fluorescence emission is nearly constant over the first several cycles, appearing as a baseline or plateau in the above figures. As thermal cycling continues, an increase in the amount of fluorescence emission above baseline can be expected to be observed, indicating that amplified products (or depleted probes) have accumulated until the fluorescence emission of one probe in the presence of the amplified products (or depleted probes) exceeds the detection threshold of the PCR device. The exponential phase is entered since the exponential increase in fluorescence emission occurs, and finally reaches another plateau when one of the components in the PCR mixture becomes limited. The above plot typically results in a sigmoidal curve with a plateau at each end and an exponential phase in the middle. In the exponential phase, the amount of fluorescence emission of the probe increases at each cycle by a factor of (1+ E) of the previous amount, where E is the efficiency of amplification and is ideally 100% or 1. It is well known that the greater the initial amount of nucleic acid template used to amplify a product, the earlier an increase above baseline is observed. As is well known in the art, the above-described plot of fluorescence emission versus cycling provides important information to know the initial copy number or amount of a nucleic acid template.

As is known in the art of real-time PCR, an unknown amount of a nucleic acid template can be quantified by comparing a plot of fluorescence emission versus cycle of the nucleic acid template to a normalized plot.

In one embodiment of the invention, when a plurality of nucleic acid templates are amplified to obtain a plurality of amplification products, each product is preferably compared to a standard curve obtained using the same product. A standard curve can be obtained with each dilution of each PCR mix to produce a separate product. Preferably, at each dilution, multiple products are included in a single PCR mixture and the fluorescence emission reading for each probe can be measured and plotted to generate a standard curve, based on the methods described in the present invention.

The initial amount of a nucleic acid template in a sample can also be determined without using a standard curve by normalizing the template to a calibrator or a homogenizer molecule. For example, the GAPDH and b-actin genes are common housekeeping genes, and can be used as calibrator templates due to their abundant presence and stability. The sample for the homogenizate may be cells, tissues or organs that have not been processed.

In one embodiment of the invention, multiple nucleic acid templates of interest are amplified and quantified in a single PCR mixture. The initial amount of each nucleic acid template can be calculated simultaneously and normalized to one kind of homogenizer. And it is expected that multiple nucleic acid templates and one homogenous molecule template can be monitored and amplified in the same PCR reaction. It is further contemplated that more than one housekeeping gene template or homogenizate may be amplified in the same PCR reaction with multiple nucleic acid templates. It is further contemplated that the relative amounts between these templates or the ratio between these templates may be determined from a single PCR mixture.

Step (c) comprises measuring the melting profile of the double stranded portion between the first and second oligonucleotides of the probes by detecting signals from the labels on the unconsumed probes as a function of temperature at least once,

To determine the melting (profile) curve of each probe or group of probes, the reaction mixture is irradiated with light that is absorbed by the labels on the probes, and the fluorescence of the reaction is monitored as a function of temperature. More specifically, the fluorescence of the label is measured as the temperature of the sample increases (or decreases) until the fluorescence reaches a baseline level.

The data may be plotted as fluorescence versus temperature, or as fluorescenceThe first derivative is shown plotted against temperature. The two maps can be switched over, but each focuses the viewer's attention on a different aspect of the data. Derivative maps are best suited to observe melting peaks (or T)_m). Whereas in the fluorescence vs. temperature diagram, broadening of the transition and the appearance of low melting point transitions are more easily observed. By observing a plot of the first derivative of fluorescence versus temperature, the point at which the rate of fluorescence increase or decrease transitions can be more easily identified. The point at which the rate of change in fluorescence is greatest is considered to be the melting temperature of the probe duplex. If a probe has a higher T_mIt forms a probe duplex that is stronger and melts at a correspondingly higher temperature than the other probes. The distinct melting temperatures of the different probes allow identification of which probes are consumed during amplification (assuming that the probes have the same label).

In some methods of the invention, fluorescence is monitored as a function of a denaturing gradient. However, what is actually monitored is the change in fluorescence resulting from dissociation of the two strands of the double-stranded portion of the probe, regardless of the type of gradient. The denaturing gradient may be a thermal gradient. The invention is also directed to a method, characterized in that temperature-dependent fluorescence is detected during or after amplification, preferably by PCR. However, it is generally advisable if the monitoring of the temperature-dependent fluorescence is part of a homogeneous detection regime, so that the (PCR) amplification and the monitoring of the temperature-dependent fluorescence take place in the same reaction tube without opening the reaction chamber in the middle.

Melting profile analysis can be achieved by detecting temperature dependent fluorescence during melting or hybridization. Typically, melting curve analysis is performed as slowly as possible to produce accurate and highly repeatable data to achieve accurate determination of the melting point, which is defined as the maximum of the first derivative of the temperature vs. fluorescence plot. However, certain benefits may also be realized if the selected time parameter is relatively short.

The measurement of the melting profile may be performed after amplification is complete (post-amplification melting profile) and/or at each cycle or selected cycles during amplification (in-amplification melting profile).

In one embodiment, temperature dependent fluorescence is monitored after completion of a PCR reaction. In an alternative embodiment, temperature dependent fluorescence is monitored in real time during a PCR reaction.

In one embodiment, the amplification product (amplicon) remains double stranded during the course of the measurement and thus is not involved in a detectable change in signal or is little if any involved. In yet another embodiment, the measurement of the melting profile may be performed when some of the amplification products are in single stranded form.

The skilled person will be able to determine whether at least one target nucleic acid has been amplified from the post-amplification melt profile alone (and without comparing the melt profile with any other melt profile). For example, FIGS. 1 and 2 show the "flatter" curve obtained after amplification, unlike the more "S" shaped curve characteristic of an unconsumed probe.

The method of the present invention may further comprise the step (d)

(i) Comparing at least two melt profiles obtained in step (c)

And/or

(ii) Combining a melting profile obtained in step (c) with

A previously obtained melting profile of the same probe, or

A theoretical melting profile of the same probe,

make a comparison

The melting profile can be measured before amplification occurs (pre-amplification melting profile), and/or after amplification is complete (post-amplification melting profile), and/or during each cycle or selected cycles (in-amplification melting profile).

The pre-amplification melt profile of the probe may be measured in the same reaction tube prior to initiation of amplification, or may be measured in another reaction tube in which amplification does not occur due to the absence of one or more components of the reaction mixture necessary for amplification. Examples of such components include a dNTP, a polymerase and a target nucleic acid.

It should be noted that the method of the invention does not necessarily require the measurement of the pre-amplification melt profile as described above as part of the method of the invention. This profile may be a profile characteristic of the probe or combination of probes involved and may have been previously measured and/or stored in a recoverable manner, for example in a computer-readable form.

Each probe in the reaction mixture is specific for a particular target nucleic acid. The different probes used are labeled with the same label or labels whose emission spectra are indistinguishable, and are selected to have different melting characteristics, e.g., different melting profiles or different melting temperatures (Tmelting)_m) In (1).

The melting profile obtained in step (c) is a composite of the melting profiles of the various probes present in the amplification reaction mixture. Due to the fact that different probes are selected to have different melt profiles, the skilled person can distinguish the individual contributions of each probe to the combined melt profile by manual methods or preferably by methods using a computer. In this way, the presence or absence of a particular probe, and therefore the presence or absence of a particular target nucleic acid in a sample, can be distinguished from one another.

Comparison of the pre-amplification and post-amplification melting profiles for each probe identifies which feature of the curve that is characteristic of a particular probe has disappeared or is diminished, thereby indicating that the particular probe has been consumed during amplification and further indicating that the corresponding target nucleic acid is present in the sample.

Preferably, the comparison between the melting profiles of the probe before amplification is performed and after amplification is complete or after amplification is performed for a number of cycles is performed by a computer program. The computer program is able to determine which probe or how much of it is consumed, which in turn indicates which target nucleic acid or how much of the corresponding target nucleic acid is present in a sample.

Melting temperature (T) of probes having the same label or different labels whose emission spectra are indistinguishable_m) The melting temperatures of similarly labeled probes are generally different from each other. In one embodiment, the plurality of probes in a probe set are each labeled with the same label (or different labels whose emission spectra are indistinguishable), and each probe has a unique melting temperature range. In a multiplex assay, the lowest T is observed when the reaction temperature is increased from the hybridization temperature to a denaturation temperature_mThe probe duplex of (a) melts first, with a second lowest T_mThe probe duplex of (a) is then separated, with the highest T_mThe probe duplex of (3) is finally denatured. At the same time, due to the gradual melting of the probe duplex, the fluorescence emission of the label associated with the probe undergoes a change proportional to the gradually increasing reaction temperature, allowing each probe to be distinguished in the combined melting profile. The shape and position of the melting curve is a function of the GC/AT ratio, length and sequence of the double stranded portion of the probe.

Preferably, T of probes having the same label or different labels whose emission spectra are indistinguishable_mT of similarly labeled probe to others_mThere is a difference of at least 2 ℃, preferably at least 3 ℃,4 ℃ or 5 ℃.

In one aspect of the invention, real-time fluorescence monitoring of a PCR reaction is used to obtain a melting curve of a probe during the PCR reaction. The temperature cycling that drives the amplification in PCR alternately denatures the accumulating product and the labeled probe at a higher temperature and anneals at least one strand of the probe and the primer to the product at a lower temperature, thereby allowing some of the probe to be consumed. As the sample is heated, the internal double-stranded portion of the remaining (unconsumed) probe gradually melts, and the fluorescence during heating through the melting temperature of this internal double-stranded portion is plotted as a function of temperature, yielding a melting curve for the probe. Thus, continuous monitoring of fluorescence during a PCR reaction provides a system for detecting changes in probe concentration via probe melt profiles. Such a system, in particular a HRM-PCR system, can be used to distinguish the remaining probes by a difference in melting temperature of less than 2 ℃.

The present invention also provides a method for monitoring one PCR amplification of at least two target nucleic acids, the method comprising:

(i) one or more pairs of forward/reverse oligonucleotide primers, wherein said primer pairs amplify one or more target nucleic acids when present in said sample, and a nucleic acid polymerase,

(ii) a set of probes comprising two or more probes, wherein at least one probe comprises a double-stranded portion, which may be a molecular beacon probe, or which may comprise two strands:

a first oligonucleotide comprising a first region substantially complementary to a portion of a target nucleic acid and a second region, and

at least one second oligonucleotide comprising a region sufficiently complementary to a second region of said first oligonucleotide,

wherein each probe comprises a detectable label capable of producing a variable signal reflecting the presence or absence of a double stranded portion formed between the first and second oligonucleotides of that probe, and

wherein at least two of said probes comprise the same detectable label or different detectable labels whose emission spectra are indistinguishable,

wherein the melting characteristics of the double-stranded portion of each of said probes are different;

Wherein, when a target nucleic acid is present, the corresponding probe, which is sufficiently complementary to a portion of the target nucleic acid, is consumed during the amplification reaction; and is

Wherein step (b) further comprises step (b 1): acquiring Fluorescence Emission (FE) cycle by cycle at a plurality of detection temperatures (MT), wherein the Fluorescence Emission (FE) is baseline corrected fluorescence (dR),

wherein when the amplification reaction mixture comprises "n" probes for multiplex detection of "n" target nucleic acids, the first probe has a melting temperature T_m1, the second probe has a melting temperature T_m2, the melting temperature of the third probe is T_m3, the melting temperature of the nth probe is T_mn, wherein T_m1＞T_m2＞T_m3…＞T_mn, wherein the percentage of the double-stranded form of each probe at a particular temperature or different temperatures is determined experimentally or calculated theoretically by a computer program, wherein a first fluorescence emission FEa is obtained at a measurement temperature MTa, wherein more than 50% of the first probe is in double-stranded form at the MTa, and a second fluorescence is obtained at a measurement temperature MTbLight emission FEb, where more than 50% of the second probe at MTb is in double-stranded form, fluorescence emission FE (n-1) is obtained at a measurement temperature MT (n-1), where more than 50% of the n-1 probes at MT (n-1) are in double-stranded form, fluorescence emission FEn is obtained at a measurement temperature MTn, where more than 80% of the n-th probe at MTn is in double-stranded form, alternatively, fluorescence emission FE0 is obtained at a measurement temperature MT0, where no more than 10% of the first probe at MT0 is in double-stranded form, where n is a positive integer and n.gtoreq.2,

wherein step (b) may further comprise step (b 2): for each probe, the actual consumption of fluorescence emission from the consumed probe was determined cycle by cycle, where the actual consumption of fluorescence emission of the kth probe was recorded as ACA_k. At a specific measurement temperature (MTa), where a certain percentage (dska)% of one probe is in ds (double stranded) form, the first probe contributes (ds1 a)% (ACA) to the fluorescence emission FE at the measurement temperature MTa₁) The contribution of the second probe was (ds2 a)% (ACA)₂) The contribution of the kth probe was (dska)% (ACA)_k). For example, at 60 ℃, 70% of probe 1 is in the ds form; at 50 ℃, 80% is in the ds form. The contribution of probe 1 to FE at 60 ℃ was 70% (ACA)₁) (ii) a The contribution of probe 1 to FE at 50 ℃ was 80% (ACA)₁). If there are multiple probes, FE is the total amount contributed by all probes consumed. The calculation of the Actual Consumption (ACA) can be performed with the following formula:

at a temperature "a", the total fluorescence emission is

FEa＝(ACA1)*(ds1a)％+(ACA2)*(ds2a)％+(ACA3)*(ds3a)％…+(ACAn)*(dsna)％

At a temperature "b", the total fluorescence emission is

FEb＝(ACA1)*(ds1b)％+(ACA2)*(ds2b)％+(ACA3)*(ds3b)％…+(ACAn)*(dsna)％

At a temperature "c", the total fluorescence emission is

FEc＝(ACA1)*(ds1c)％+(ACA2)*(ds2c)％+(ACA3)*(ds3c)％…+(ACAn)*(dsna)％

And so on. The ACA alone can be calculated from the above formula.

Wherein the amount of fluorescence emission of each probe is derived by a computer program or manually.

The invention also provides a computer software product for use with the method of the invention, which software is adapted to compare the melting profiles of probes and/or to quantify a real-time PCR amplification comprising multiple targets, when run in a suitable data processing method, and which software enables the calculation of fluorescence emission and Actual Consumption (ACA).

Generally, ACA can be calculated manually once the fluorescence emission values are obtained by the PCR equipment and the percentage of the double-stranded form of each probe at each temperature is known. However, it is often desirable to automate the above calculations by using a computer system.

In a further embodiment, the invention relates to a computer architecture comprising a computer memory storing a computer software program, wherein said computer software program, when executed by a processor or in a computer, performs the method according to the invention. In a preferred embodiment, a computer program product comprises a computer memory storing a computer software program, wherein said computer software program runs a method comprising the steps of calculating ACA and/or determining a feature of a melt map during or at the end of amplification.

As will be appreciated by those skilled in the art, a computer program product of the present invention, or a computer software program of the present invention, may be stored and/or run on a PCR apparatus and used to calculate the amount of each probe.

The present invention further provides a kit for detecting one or more target nucleic acids, the kit comprising a probe comprising:

a first oligonucleotide consisting of 15 to 150 nucleotides comprising a first region substantially complementary to a portion of a target nucleic acid and a second region, and

at least one second oligonucleotide of 4 to 150 nucleotides, said second oligonucleotide comprising a region substantially complementary to said second region of said first oligonucleotide,

wherein each probe comprises a detectable label or combination of detectable labels capable of producing a variable signal reflecting the presence or absence of a double stranded portion of the first and second oligonucleotides of that probe,

and wherein

(a) The first oligonucleotide of said probe does not comprise a label and said second oligonucleotide comprises a first label and a second label, wherein said first label is associated with said second oligonucleotide at or near one end thereof and said second label is associated with said second oligonucleotide at or near the other end thereof, whereby when the second oligonucleotide is not hybridized to the first oligonucleotide, the second oligonucleotide is in a random coil configuration or a stem-loop configuration that draws the first and second labels into close proximity, and wherein when said second oligonucleotide is hybridized to said first oligonucleotide, the two labels are remote from each other; or

(b) Said first oligonucleotide comprising no label and said second oligonucleotide comprising a label, wherein detectable signal emission from said label is capable of being altered relative to emission of the label in single stranded form from said second oligonucleotide when said second oligonucleotide is hybridized to said first oligonucleotide to form a double stranded portion of said probe; or

(c) The first oligonucleotide of the probe is free of a label and the probe comprises two second oligonucleotides capable of hybridizing adjacent or sufficiently adjacent to different moieties on a second region of the first oligonucleotide, wherein one of the second oligonucleotides is associated with a first label and the other second oligonucleotide is associated with a second label, such that when the two second oligonucleotides hybridize to the first oligonucleotide, the labels are drawn into close proximity and one label affects the signal from the other label.

The present invention also provides the use of a probe as defined in (a) to (c) above in a method of the invention.

The present invention also provides the use of a probe in a method as disclosed herein, the probe comprising:

and wherein

(a) The first label is associated with a second region of the first oligonucleotide and the second label is associated with a region of the second oligonucleotide complementary to the second region of the first oligonucleotide such that the first and second labels are drawn together into close proximity when an internal duplex of the probe is formed, or

(b) The first and second oligonucleotides of the probe are linked by a linking moiety comprising a chemical moiety that is either a nucleotide or a non-nucleotide, thereby allowing the first and second oligonucleotides to form a stem-loop structure, wherein the first and second oligonucleotides are labeled separately, such that when the probe forms an internal stem-loop structure, the labels are pulled into close proximity and one label affects the signal from the other label.

Another aspect of the invention is directed to a method of detecting one or more variant nucleotides in a target nucleic acid from a sample, the method comprising:

(a) contacting a sample comprising target nucleic acid with an amplification reaction mixture comprising:

(ii) at least one probe pair, wherein a first probe of the probe pair comprises a sequence complementary to a wild-type target nucleic acid sequence (normal sequence) and a second probe of the probe pair comprises a sequence complementary to a target nucleic acid sequence comprising a variant nucleotide (e.g., a SNP, a mutated nucleotide, etc.), wherein each probe of the probe pair comprises:

wherein each probe in the pair of probes comprises the same second oligonucleotide,

wherein each probe comprises a detectable label or combination of detectable labels capable of producing a variable signal reflecting the presence or absence of a double stranded portion of the first and second oligonucleotides of that probe, and

wherein at least two of said probes comprise the same detectable label or different detectable labels whose emission spectra are indistinguishable, and wherein the double-stranded portion of the first and second oligonucleotides of each of said probes has a different melting characteristic;

Wherein, when a target nucleic acid is present, a first oligonucleotide of the probe that is sufficiently complementary to a portion of the target nucleic acid is consumed during the amplification reaction; and is

(c) Measuring the melting profile of any double stranded portion formed between the first and second oligonucleotides of any unconsumed probes by detecting the signal from the label on these probes as a function of temperature at least once,

The same second oligonucleotide in the probe pair may comprise a universal base or inosine corresponding to a variant nucleotide in the target nucleic acid sequence. The universal base can be 3-nitropyrrole 2' -deoxynucleoside, 5-nitroindole, pyrimidine analogue or purine analogue. Inosine naturally occurs at the wobble position of certain anticodons of transit RNAs and is known to form base pairs with A, C and U during translation (fig. 17).

To scan for multiple mutations or SNPs in a target sequence, multiple first oligonucleotides of different probes that hybridize to different sites on the same amplification product can be included in a single reaction. The probes may comprise a competitive pair of probes, the first probe of the pair hybridizing to a wild-type (normal nucleotide) sequence; the second probe of the pair hybridizes to a target sequence that includes a variant (mutated) nucleotide. When a wild-type target sequence is present, the probe complementary to the wild-type target sequence is consumed. When a target sequence containing a variant nucleotide exists, a probe complementary to the variant target sequence is consumed. The plurality of first oligonucleotides can hybridize to the same strand of the target nucleic acid sequence adjacent to each other, or with some overlap between adjacent first oligonucleotides (FIG. 17).

Another aspect of the invention is a primer and probe combination for monitoring, detecting or quantifying a plurality of nucleic acid substrates in an amplification reaction. The amplification reaction is based on the melting characteristics of the primer or probe hybridized to the primer amplification product (FIG. 14).

The primer and probe combination may contain a first primer, a second primer and a first probe (FIG. 14A).

The second primer herein contains, in the 3 'to 5' direction, a3 'target-specific promoter region which is relatively complementary to a target nucleic acid sequence, a probe-complementary region which contains a sequence complementary to the first probe, and a 5' consensus region which contains a sequence identical or relatively identical to the first primer.

Where the first probe hybridizable to the second primer comprises a first label and the first primer comprises a second label, the first label and the second label are capable of interacting when they are in close proximity.

Where the first primer is extendable on a template that is complementary to the extended strand of the second primer.

Here, when the first probe is hybridized to the extension product of the first primer, the two labels are drawn closer together and a detectable signal is generated.

In one embodiment, the primer and probe combination, the first primer is a consensus primer, the first probe is a consensus probe, and the second primer comprises a primer set, wherein each primer in the primer set has an identical label. In the second primer set, a 3' target-specific priming region can hybridize to each target sequence, the first probe hybridizes to the second primer or to the extended strand of the first primer, and the Tm or melting profile of the hybridization is specific for each target sequence. This first probe is resolvable with respect to the different Tm or melting profile of each target, such that the Tm or melting profile provides information on the presence or absence of the target (fig. 14B).

In another case, the probe-complementary region of the second primer and the 3' target-specific initiation region do not overlap, where the probe-complementary region contains a sequence that may be unrelated to the target sequence.

In yet another case, the probe-complementary region of the second primer overlaps the 3 'target-specific region, or is contained within the 3' target-specific priming region (FIG. 14A), and the Tm for hybridization of the second primer to the target sequence is higher than for the second primer to the first probe, so that the second primer forms a stronger hybridization product with the target when the target sequence is present.

One of the labels may be a quencher or fluorescein, and the first probe hybridises to the extended strand of the first primer, which may bring the two labels closer together, resulting in a tight quenching or FRET effect.

Another aspect of the invention is a method for monitoring, detecting or quantifying a series of nucleic acid substrates in an amplification reaction.

The method for detecting or quantifying a target nucleic acid comprises: (a) treating the nucleic acid sample with a combination of primers and probes under amplification reaction conditions, the second primer hybridizing to the target nucleic acid and extending, the extended strand of the second primer separating from the template and participating in the next round of hybridization, extension,

here, the concentration of the second primer is lower than that of the first primer, and the first primer participates in the amplification reaction as the second primer is gradually consumed; (b) detecting signal generation from the extension product of the first primer hybridized to each cycle from the first probe; (c) detecting a melting profile of the hybridized duplex of the first probe and the first primer extension product, wherein the melting profile is determined by exciting the reaction mixture and monitoring fluorescence as a function of temperature, a characteristic Tm or melting profile indicating the presence of the target sequence.

Preferably, the amplification reaction is asymmetric amplification, in which the extended single strand of the first primer gradually accumulates during amplification, which can be achieved by unequal ratios of the forward primers.

In yet another aspect of the invention, a kit for monitoring, detecting or quantifying a plurality of nucleic acid substrates in an amplification reaction, a kit for detecting or quantifying a target nucleic acid comprising, a combination of a primer and a probe, or a combination of a set of primers and a probe, the combination of primers and probes can comprise a first primer, a second primer and a first probe.

The first probe capable of hybridizing to the first primer extension strand may contain a target specific sequence or a random sequence and may be of any length.

Generally, the first probe is 4 to 60 nucleotides, preferably 5 to 25 nucleotides in length.

The probe-complementary region of the second primer comprises a sequence capable of hybridizing to the first probe and is designed such that the hybridized duplex of the probe-complementary region and the first probe has a unique Tm or melting profile relative to each target sequence.

The 5 'consensus region of the second primer contains a sequence that is the same as or similar to the 3' promoter region of the first primer, or the entire sequence of the first primer. Designed such that the first primer hybridizes to the template of the second primer replication product and initiates extension.

In one instance, the first primer is a target-specific primer comprising a 3' promoter region that is complementary to the target. Here, the first probe in the reaction contains a sequence that hybridizes to the extended strand of the first primer and is adjacent to the first primer.

In a preferred aspect, the first primer is a consensus primer, the first probe is a consensus probe, and the second primer comprises a primer set, wherein the 3' target-specific priming region of each second primer of the primer set is capable of specifically hybridizing to each target sequence and priming amplification. The probe-complementary region of each second primer herein is capable of hybridizing to the first probe and is unique.

The second primer may be a type-specific primer for a nucleotide that is typing or detecting a mutation with the SNP. The terminal nucleotide of the second primer is selected to be complementary to the suspected mutant nucleotide or to the normal nucleotide. Thus, when the second primer hybridizes to a region containing a suspected nucleotide in the diagnostic region, the extension product of the second primer is synthesized, and conversely, is not synthesized. The genotype-specific second primer may differ due to the probe complementary region, which allows the melting profile to be distinguished between different genotypes.

Alternatively, the first primer and the first probe may be linked by a single linker. The ligated primers/probes are in the following order, from 5 ' to 3 ', first probe-linker-first primer, where the first probe may be referred to as the 5 ' tail region of the first primer. The linking group may be a natural nucleotide or any chemical group. The linking group may contain a blocking group. If a blocking group is present, replication of the first probe is blocked. The blocking group may be a hydrocarbon chain, a HEG non-nucleotide linkage, an abasic sugar, a nucleotide derivative or a dye.

In some cases, the second primer set used in the reaction may be nested primers. Nested primers used for amplification are oligonucleotides having complementary target sequences.

The first primer may contain a first label. When the first probe is hybridized to the extension of the first primer, hybridization/melting of the first probe generates a detectable signal or a specific melting profile. Preferably, the 3' promoter region of the first primer may comprise a second label, the first label and the second label being an interactable label pair. When the first probe hybridizes to the extension of the first primer, hybridization brings the two labels closer together, so that the melting profile of the first probe indicates whether the target sequence is present.

When the labeled primer/probe hybridizes to the extension product of the first primer, the two labels are brought into a FRET or tight quenching relationship. The label on the primer or probe may be in any position as long as they can produce an interaction.

A method for detecting a target nucleic acid in a sample comprising:

(a) providing a template nucleic acid, the template nucleic acid being derived from a target nucleic acid, the first primer being hybridizable to the template nucleic acid;

(b) treating the template nucleic acid with a first primer or a plurality of first primers, each first primer hybridizing under hybridization conditions to its corresponding target nucleic acid, each first primer comprising a3 'promoter region complementary to a first region of the template nucleic acid and a 5' tail region capable of hybridizing to an extension of the first primer.

Alternatively, the template nucleic acid is treated with a first primer or a plurality of first primers and a first probe, each first primer hybridizing to the template nucleic acid under hybridizing conditions, each first primer comprising a 3' promoter region complementary to a first region of the template nucleic acid, the first probe capable of hybridizing to an extension of the first primer.

(c) Maintaining the mixture of step (a) in extension conditions comprising the corresponding nucleoside triphosphates and a DNA polymerase, under which conditions the annealed first primer is extended and an extension product is synthesized.

(d) The extension product is isolated from the template.

(e) Maintaining the reaction of step (c) under conditions comprising a buffer, a corresponding temperature or a series of temperatures wherein the 5' tail region of the first probe or first primer hybridizes to the extension portion of the first primer. Hybridization or melting of the first probe produces a detectable signal or characteristic melting profile.

The method may further comprise repeating steps (a) and (e) in one amplification reaction. Any amplification system can be used containing these steps like PCR, SDA, LAMP, 3 SR. PCR is the preferred amplification method containing these steps.

In the methods described above, a sample is provided that is suspected of containing a target nucleic acid or a mutant nucleotide of interest. The target nucleic acid in the sample may comprise double stranded gDNA or cDNA the DNA may be denatured by any method, including known physical, chemical or enzymatic treatments. The preferred physical treatment is denaturation of DNA by heating to 99% or more. Typical heat treatment temperatures are 80 ℃ to 105 ℃ and times can be several seconds or minutes. In addition, if the target nucleic acid is inherently single-stranded, such as a single-stranded RNA or DNA virus, a denaturation step is not required.

The deformed nucleic acid strand is mixed with an oligonucleotide primer under hybridization conditions that allow the primer to bind to the single-stranded nucleic acid. In one instance, the template nucleic acid is provided as a denatured target nucleic acid and is capable of hybridizing to the first primer. The first primer is a target nucleic acid specific primer, each primer in the primer set comprising a 3' priming region capable of hybridizing to each target sequence and initiating extension. For example, each first primer contains a 5' tail region that hybridizes to an extension of the first primer. In another example, the first primer does not contain a 5' tail region, but the reaction contains a first probe that hybridizes to the extension of the first primer and is adjacent to the first primer.

The annealed first primer is extended by the action of a polymerase. Template-dependent primer extension is catalyzed by a polymerase enzyme and has a sufficient amount of deoxynucleoside triphosphates (dATP, dGTP, dCTP, and dTTP) or the like, containing the corresponding salt, metal cation, and pH buffering system in the reaction medium. Suitable polymerizing agents are enzymes known to catalyze template nucleic acid-dependent DNA synthesis. Reaction conditions capable of catalyzing DNA synthesis are known.

The extended product is separated from the template by heating, e.g., the extended product can be isolated at 95 ℃. Alternatively, the extended product may be isolated in a strand displacement manner.

After the product of the first primer is separated from the template, the reaction mixture is maintained under reaction conditions comprising a buffer and a corresponding temperature or a series of temperatures. Where the 5' tail region of the first probe or first primer hybridizes or melts to the extension of the first primer. This process produces a detectable signal or characteristic melting spectrum.

The determination of Tm of a double-stranded structure of an amplification product or a probe or the monitoring of a melting curve has been widely used. In this invention, detection of multiple target nucleic acids can be achieved by hybridization/melting spectrum analysis of the label pair and the first probe hybridized to the extension portion of the first primer.

In another case, the first primer is a consensus primer. The first probe is a common probe. Multiple target nucleic acids can be detected by melting profile analysis of the target nucleic acids by common probe hybridization and performed in one detection channel.

Here, the step (a) of providing a template nucleic acid comprises: i) treating the sample nucleic acid with a second primer, the second primer being a target-specific primer set. Under hybridizing conditions, each second primer anneals to the target nucleic acid. The second primer is composed of, in the 3 'to 5' direction, a target-specific portion that is substantially complementary to the target nucleic acid, a tail probe-complementary portion that hybridizes sequentially to the first probe or the tail of the first primer, and an identical or similar 5 'consensus portion that is the 3' promoter region of the first primer; ii) maintaining the reaction solution in extension conditions comprising the respective nucleoside triphosphates and a nucleic acid polymerase for extending the annealed second primer to synthesize an extension product; iii) separating the extension product from the template; iv) annealing the amplification product single-stranded above the other amplification primer (third primer) or the third primer set, and extending the annealed third primer; and v) denaturing the double stranded extension product into single stranded form, including the template nucleic acid, such that the single stranded template nucleic acid is capable of hybridizing to the first primer (FIG. 15).

This third primer may be one of the amplification products. The amplification products are selected based on their relative positions in the duplex sequence such that the extension product of one amplification primer, when separated from the template (complementary strand), serves as a template for extension of the other primer.

The concentration of the common first primer present in one reaction is at least 3 times the concentration of the second primer. Preferably, the concentration of the common first primer present in one reaction is at least 6 times the concentration of the second primer.

Drawings

FIG. 1 is a schematic representation of the melting profiles of a nucleic acid probe of the invention, consumed during amplification, before and after amplification.

FIG. 2 is a schematic representation of the melting profiles of a mixture of the nucleic acid probes of the invention (probes 1 and 2), one or both of which are consumed in amplification, before and after amplification.

FIG. 3 illustrates a real-time measurement of the synthesis of one amplicon at different temperatures.

FIG. 4 shows examples of different probes that can be used in the present invention.

FIG. 5 shows an example of a method of the invention in which a first oligonucleotide of a probe is hybridized to a target nucleic acid sequence.

FIG. 6 shows an example of a method of the invention in which a first oligonucleotide of the probe is incorporated into the amplification product.

FIG. 7 shows an example of a method of the invention in which a first oligonucleotide of the probe is extended and degraded during amplification.

FIG. 8 shows an example of a method of the invention in which the first oligonucleotide of the probe is degraded during amplification.

FIG. 9A shows a melting profile of probes 1 and 2 of the present invention before amplification. FIG. 9B shows a melting profile of a mixture of probes 1 and 2 at different ratios.

FIG. 10 is a graph showing a melting profile of the mixture of the present probes 1 and 2 in 4 reactions before amplification.

FIG. 11 illustrates one real-time measurement of amplicon synthesis at different temperatures in the example.

FIG. 12 shows a melting profile of a mixture of the present probes 1 and 2 in 4 reactions after amplification.

FIG. 13 shows an amplification plot in which the amount of fluorescence emitted from the first probe (K10) is labeled as FE 1. The calculated FE1 × P map is denoted as FE1 xP. An amplification chart of the fluorescence emission amount of the second probe (SV40) is shown as FE2- (FE 1x P).

FIG. 14 shows a combination of primers and probes (A) and a combination of a set of primers and probes (B).

FIG. 15 is a schematic representation of a method for detecting and quantifying 3 target nucleic acids using a set of primer and probe combinations.

FIG. 16 is a schematic representation of an improved primer and probe combination, the primer and probe being linked by a linker.

FIG. 17 set of Fam-labeled probes comprising sequences complementary to wild-type sequences, the set of probes having different T' s_m(ii) a Another set of probes labeled with Hex comprising sequences complementary to variant sequences of the same target nucleic acid sequence.

FIG. 18 shows a method of using the probe set described in FIG. 14.

FIG. 19 shows the results of an example of triple amplification. Having different T_mAre labeled with Fam dyes. (A) Showing a melting pattern generated in a tube to which the target DNA was not added. The following figure shows a comparison between the melting pattern produced in a tube to which the target DNA was not added and the melting pattern produced in a tube to which the target DNA was added. (B) A melt map generated in a tube containing a sample in which the target 2 is present. (C) A melt map generated in a tube containing a sample in which the target 3 is present. (D) A melt map generated in a tube containing a sample in which targets 2 and 3 are present. (E) A melt map generated in a tube containing a sample in which target 1 is present. (F) A melt map generated in a tube containing a sample in which targets 1 and 2 are present. (G) A melt map generated in a tube containing a sample in which targets 1 and 3 are present. (H) A melt map generated in a tube containing a sample in which targets 1, 2 and 3 are present.

FIG. 20(A) shows the melting temperature and signal collection parameter settings. (B) Is the melting profile of probe 1, probe 2 and the mixture of probes 1 and 2. (C) A multivariate view showing how the melting curve is estimated as a percentage of the double-stranded form of one probe.

FIG. 21 is a graph showing an amplification chart of actual consumption and a standard curve (example 4).

FIG. 22 shows the results of one experimental example of quadruple amplification: various combinations of target nucleic acids are used in the reaction, reaction A being template-free; reaction B contains a target 3 template; reaction C contains target 2 template; reaction D contains target 4 template; reaction E contains target 1 and target 3 templates; reaction F contained target 1, 3 and 4 templates.

Detailed Description

The invention is further illustrated in the following examples in which reference is made to parts and percentages by weight and temperatures in degrees celsius unless otherwise indicated. It should be understood that these examples, while representing preferred embodiments of the invention, are provided by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

The disclosure of each reference cited herein is incorporated by reference in its entirety.

Example 1

All primers used in the following experiments were synthesized by EUROGENTEC.

The amplification primers and probes were as follows:

K10R266Fam GttcaATTGGGTTTCACCGCGCTTAGTTACA(SEQ ID NO：1)；

K10R266Dab GCGCGGTGAAACCCAATTGAAC(SEQ ID NO：2)；

SV40R1F3FAM ATCAGCCATACCACATTTGTAGAGGTTTTAC(SEQ ID NO：3)；

SV40R1F3Dab CAAATGTGGTATGGCTGAT(SEQ ID NO：4)；

K10F155 CTCTGCTGACTTCAAAACGAGAAGAG(SEQ ID NO：5)；

SV40RealR CCATTATAAGCTGCAATAAACAAGTTAACAAC(SEQ ID NO：6)

primer/probe K10R266Fam (first oligonucleotide) was labeled with FAM at its 5' end. K10R266Dab (second oligonucleotide) comprises DABCYL at its 3' end. K10R266Fam and K10R266Dab are capable of forming a double stranded portion as follows:

Fam-5′GTTCAATTGGGTTTCACCGCGCTTAGTTACA3′(SEQ ID NO：1)

DABCYL-3′CAAGTTAACCCAAAGTGGCGCG5′(SEQ ID NO：2)

this hybrid was referred to as probe K10R 266. The primer/probe SV40R1F3FAM (first oligonucleotide) was labeled at its 5' end with FAM. SV40R1F3Dab (second oligonucleotide) comprises DABCYL at its 3' end. The SV40R1F3FAM and SV40R1F3Dab are capable of forming a double stranded portion as follows:

Fam-5′ATCAGCCATACCACATTTGTAGAGGTTTTAC3′(SEQ ID NO：3)

DABCYL-3′TAGTCGGTATGGTGTAAAC5′(SEQ ID NO：4)

this hybrid was designated as probe SV40R1F 3.

The first and second oligonucleotides described above are combined in various ratios, typically 1: 3, to form a partially double-stranded linear DNA probe. The formation of the first and second oligonucleotide hybrids brings the quencher and the fluorescent group into close proximity when the target nucleic acid is absent, effectively quenching the fluorescent signal. When the target nucleic acid is present, the first oligonucleotide preferentially hybridizes to the target sequence and is incorporated into the amplicon. As a result, the quencher is separated from the fluorescent group, resulting in an enhancement of fluorescence emission.

Primer pair K10R266Fam and K10F155 amplified a 110bp product in the presence of a K10 target sequence. SV40R1F3FAM and SV40RealR amplified a 125bp product in the presence of an SV40 target sequence.

Example 2

The melt profile experiments were performed as follows: the thermal stability of the probe was verified in a melt map experiment where fluorescence emission was measured at temperatures ranging from 40 to 90 ℃. Melting temperature T_mIs defined as the characteristic temperature at which the first and second oligonucleotide duplexes melt.

The measurement of each melting profile was carried out in a reaction tube containing a 20. mu.l system of PCR buffer (1XThermoPol reaction buffer, New England BioLabs). Thermal cycling was performed in an Mx3005p quantitative PCR system (Stratagene) under the following cycling conditions: denaturation at 90 ℃ for 3 min, 1 cycle; the temperature range from 40 to 90 ℃ was held for 30 seconds, with 1 ℃ ramp per cycle, 50 cycles. Fluorescence measurements were recorded during each 30 second hold of the temperature for the 50 cycles described above.

The melting profiles of probe K10R266 (probe 1) and probe SV40R1F3 (probe 2) were determined by monitoring fluorescence at temperatures ranging from 90 to 40 ℃. K10R266 has a T of 72 DEG C_m(ii) a SV40R1F3 has a T of 62 ℃_m(FIG. 9A).

The melting profile was determined for a series of mixtures of probe K10R266 and probe SV40R1F 3:

sample 1 contained 0.5. mu.M of K10R266 and 0.5. mu.M of SV40R1F 3.

Sample 2 contained 0.5. mu.M of K10R266 and 0.25. mu.M of SV40R1F 3.

Sample 3 contained 0.5. mu.M of K10R266 and 0.125. mu.M of SV40R1F 3.

Sample 4 contained 0.5. mu.M of K10R266 and 0.0625. mu.M of SV40R1F 3.

Sample 5 contained 0.5. mu.M K10R266 and 0.003125. mu.M SV40R1F 3.

The melting map is shown in FIG. 9B.

A combination of probes K10R266 and SV40R1F3 was tested using real-time PCR analysis with plasmid DNA containing the sequences K10 and SV40 as templates.

A reaction premix containing 0.5. mu.M of K10R266 and 0.5. mu.M of SV40R1F3 (the combination of the first and second oligonucleotides were mixed in a ratio of 1: 3) and standard PCR components (NEB) was prepared. The amplification reaction was performed in a Stratagene Mx3005p quantitative PCR system under the following cycling conditions:

1. before amplification, melting profile: the temperature range from 90 to 40 ℃ is maintained for 30 seconds, decreasing by 1 ℃ with each cycle, 50 cycles, and fluorescence measurements are recorded during each 30 seconds of the 50 cycles holding the temperature.

2. Amplification: 30 cycles of 94 ℃ for 20 seconds, 63 ℃ for 30 seconds, 51 ℃ for 30 seconds, 72 ℃ for 30 seconds; the fluorescence measurements were recorded at reading steps of 63 ℃,51 ℃ and 72 ℃.

3. Melting map after amplification: the temperature range from 40 to 90 ℃ was held for 30 seconds, with each cycle increasing by 1 ℃, 50 cycles, and fluorescence measurements were recorded during each 30 seconds of the 50 cycles holding the temperature.

Four reactions were performed: reaction 1 contains a K10 template; reaction 2 contained an SV40 template; reaction 3 contained K10 and SV40 templates; reaction 4 contained no template.

The melting map before amplification of the four reactions is shown in FIG. 10.

The fluorescence emission collected at 63 ℃ during amplification is shown in FIG. 11A.

The fluorescence emission collected at 51 ℃ during amplification is shown in FIG. 11B.

The fluorescence emission collected at 72 ℃ during amplification is shown in FIG. 11C.

The post-amplification melting profiles of the four reactions are shown in FIG. 12.

The above comparison between pre-amplification and post-amplification melting profiles shows that:

in reaction 1, the K10R266 probe was consumed and the profile shows the characteristics of the SV40R1F3 probe.

In reaction 2, the SV40R1F3 probe was consumed.

In reaction 3, both the K10R266 and SV40R1F3 probes were consumed.

In reaction 4, no probe was consumed and the pre-amplification and post-amplification profiles were similar.

Cycle-by-cycle fluorescence emission FE was obtained at three measurement temperatures: MT 72 ℃,63 ℃ and 51 ℃.

Obtaining a first fluorescence emission FE1 at a measurement temperature of 63 ℃, at which no more than 10% of the second probe (SV40R1F3) is in duplex form (internal double-stranded form of the probe); the second fluorescence emission FE2 was obtained at a measurement temperature of 51 ℃ where more than 95% of both probes were in duplex form, and alternatively, a fluorescence emission FE0 was obtained at a measurement temperature of 72 ℃ where no more than 10% of the first probe (K10R266) was in duplex form.

In the above amplification reaction, there are two probes for the target sequences K10 and SV 40. At 72 ℃, 10% of the K10R266 probe was in duplex form and 0% of the SV40R1F3 probe was in duplex form. At 63 ℃, 90% of the K10R266 probe was in double-stranded form and 5% of the SV40R1F3 probe was in double-stranded form. At 51 ℃, more than 98% of all probes are in double-stranded form.

The first fluorescence emission was collected at 63 ℃ and was FE 1; the second fluorescence emission was collected at 51 ℃ and was FE 2.

FE1＝90％*ACA1+5％*ACA2

FE2＝98％*ACA1+98％*ACA2

Assuming negligible 5% ACA2, ACA1 ═ FE 1/90%; ACA2 ═ FE 2/98% -FE 1/90%. ACA1 is the actual consumption of probe 1; ACA2 is the actual consumption of probe 2.

The amplification plot of the actual consumption of the first probe (K10) is shown as ACA 1. The amplification plot of the actual consumption of the second probe (SV40R1F3) is shown as ACA2 (fig. 13).

The above calculation of the actual consumption may be performed manually. The calculation may also be performed by a computer program or software. To make amplification faster, software was designed to process fluorescence emission data from multiplex real-time PCR and perform appropriate calculations. The software also has other functions such as manual selection of Ct and subtraction of blanks.

The software described above is implemented as a plug-in to Microsoft Excel in Visual Basic for Applications (VBA). The source code is organized in two main modules. A module includes all "utility" functions, such as mathematical functions, functions to generate arrays from fluorescence emission data for display on Excel worksheets, functions to print resulting data and labels, functions to handle errors or templates, and functions to generate a specific type of chart. The second module includes functionality to control the flow of the program. This module includes all the functions needed to enable interaction with the user, such as menu selection, toolbar segmentation, and insertion/culling of data in the standard curve.

Example 3

The amplification primers and probes were as follows:

for target sequence 1(K10)

K10R266Fam GttcaATTGGGTTTCACCGCGCTTAGTTACA(SEQ ID NO：1)；

K10R266Dab GCGCGGTGAAACCCAATTGAAC(SEQ ID NO：2)；

K10F155 CTCTGCTGACTTCAAAACGAGAAGAG(SEQ ID NO：5)；

For target sequence 2(SV40)

SV40R1F3FAM ATCAGCCATACCACATTTGTAGAGGTTTTAC(SEQ ID NO：3)；

SV40R1F3Dab CAAATGTGGTATGGCTGAT(SEQ ID NO：4)；

SV40RealR CCATTATAAGCTGCAATAAACAAGTTAACAAC(SEQ ID NO：6)；

For target sequence 3(Jak2)

JKR3Fam AACAGATGCTCTGAGAAAGGCATTAGA(SEQ ID NO：11)；

JKR3FDabF CTCAGAGCATCTGTT(SEQ ID NO：12)；

JKF2 GCATCTTTATTATGGCAGAGAGAA(SEQ ID NO：13).

Oligonucleotide K10R266Fam is an amplification primer, while oligonucleotide K10R266Fam is the first oligonucleotide of Probe 1. K10R266Fam (first oligonucleotide) is labeled at its 5' end with FAM. K10R266Dab (second oligonucleotide) comprises DABCYL at its 3' end. K10R266Fam and K10R266Dab are capable of forming a double stranded portion. The hybrid formed by K10R266Fam and K10R266Dab is referred to as Probe 1.

The SV40R1F3FAM oligonucleotide is an amplification primer, and the SV40R1F3FAM oligonucleotide is the first oligonucleotide of Probe 2. SV40R1F3FAM (first oligonucleotide) is labeled at its 5' end with FAM. SV40R1F3Dab (second oligonucleotide) comprises DABCYL at its 3' end. SV40R1F3FAM and SV40R1F3Dab are capable of forming a double-stranded portion. The hybrid formed by SV40R1F3FAM and SV40R1F3Dab is referred to as probe 2.

Oligonucleotide JKR3Fam is an amplification primer, and oligonucleotide JKR3Fam is the first oligonucleotide of probe 3. JKR3Fam (first oligonucleotide) was labeled with FAM at its 5' end. JKR3FDabF (second oligonucleotide) contains DABCYL at its 3' end. JKR3Fam and JKR3FDabF were able to form a double-stranded portion. The hybrid formed by JKR3Fam and JKR3FDabF is referred to as Probe 3.

The first and second oligonucleotides described above are combined in various ratios, typically 1: 2 to 1: 4, to form a partially double-stranded linear DNA probe. The formation of the first and second oligonucleotide hybrids brings the quencher and the fluorescent group into close proximity when the target nucleic acid is absent, effectively quenching the fluorescent signal. When the target nucleic acid is present, the first oligonucleotide preferentially hybridizes to the target sequence and is incorporated into the amplicon. As a result, the quencher is separated from the fluorescent group, resulting in an enhancement of fluorescence emission.

Primer pair K10R266Fam and K10F155 amplified a 110bp product in the presence of a K10 target sequence. SV40R1F3FAM and SV40RealR amplified a 125bp product in the presence of an SV40 target sequence. Primer pair JKR3Fam and JKF2 amplified a 222bp product in the presence of Jak2 target sequence.

Melting profile analysis of probe 1, probe 2, probe 3 and mixtures of probes 1, 2 and 3 was performed using a stratagenem mx3005 real-time PCR instrument (fig. 19). The thermal parameters used were based on the parameters of the melting curve analysis software: heating at 70 ℃ for 30 seconds, cooling to 40 ℃ and keeping for 30 seconds, then slowly heating to 94 ℃, and continuously collecting fluorescence emission data during the temperature rising period. Plotting the first negative derivative of the fluorescence emission readings with respect to temperature against temperature yields a curve, each peak of which corresponds to the actual T for that probe_m。

Single, double and triple amplifications were performed using the same reaction mix, except that one target nucleic acid, two target nucleic acids or all three target nucleic acids were present.

A reaction premix containing 0.1. mu.M of each of the first oligonucleotides of the above three kinds of probes, 0.4. mu.M of each of the second oligonucleotides of the above three kinds of probes, 0.2. mu.M of each of the primers of the target nucleic acid (the first oligonucleotides other than the probes), and a standard PCR component (NEB) was prepared. The amplification reaction was performed in a Stratagene Mx3005p quantitative PCR system under the following cycling conditions:

1. amplification: 15 seconds at 94 ℃, 20 seconds at 63 ℃, 20 seconds at 50 ℃, 20 seconds at 55 ℃, 20 seconds at 63 ℃, 20 seconds at 68 ℃, 20 seconds at 72 ℃ and 40 cycles; the fluorescence measurements were recorded at reading steps of 50 ℃, 55 ℃,63 ℃, 68 ℃ and 72 ℃.

2. Melting map after amplification: after 20 seconds at 72 ℃ in the last cycle, cool to 40 ℃ and hold for 30 seconds, then slowly heat to 78 ℃ and fluorescence emission data is collected continuously during the temperature ramp.

Eight reactions were performed: reaction (a) does not comprise a template; reaction (B) comprises template target nucleic acid 2; reaction (C) comprises template target nucleic acid 3; reaction (D) comprises target nucleic acids 2 and 3; reaction (E) comprises target nucleic acid 1; reaction (F) comprises target nucleic acids 1 and 2; reaction (G) comprises target nucleic acids 1 and 3; reaction (H) comprises target nucleic acids 1, 2 and 3. The post-amplification melting profiles of the eight reactions are shown in FIG. 19.

Comparison of post-amplification melting profiles in the presence and absence of target nucleic acid revealed (FIG. 19):

in reaction A, no probe was consumed and the melting profile showed the characteristics of a mixture of all three probes.

In reaction B, probe 2 is consumed.

In reaction C, probe 3 is consumed.

In reaction D, probes 2 and 3 are consumed.

In reaction E, probe 1 is consumed.

In reaction F, probes 1 and 2 are consumed.

In reaction G, probes 1 and 3 were consumed.

In reaction H, probes 1, 2 and 3 are consumed.

Cycle-by-cycle fluorescence emission FE was obtained at five measurement temperatures: MT 50 ℃, 55 ℃,63 ℃, 68 ℃ and 72 ℃. The actual consumption of each probe can be calculated by the following formula: FEa ═ ACA1 (ds1 a)% + (ACA2) ds2 a)% + (ACA3) ds3 a)% … + (ACAn) (% dsna).

Example 4

Amplification primers for target nucleic acid 1 (K10):

K10F155 CTCTGCTGACTTCAAAACGAGAAGAG(SEQ ID NO：5)；

K10R14 CCTGAGGGTTAAATCTTCCCCATTGA(SEQ ID NO：21)

the probe for the target nucleic acid 1 (referred to as probe 1) includes: first oligonucleotide

K10R266Famph GTTCAATTGGGTTTCACCGCGCTTAGTTACA(SEQ ID NO：7)，

The 5' end of the probe is connected with Fam; the 3 'end is linked to a phosphate group other than 3' -OH; and a second oligonucleotide

K10R266Dab GCGCGGTGAAACCCAATTGAAC (SEQ ID NO: 2) with the 3' end linked to DABCYL.

Amplification primers for target nucleic acid 2(SV 40):

dsredendF2 GTAAGATCCACCGGATCTAGATAAC(SEQ ID NO：8)；

sv40testR GGGAGGTGTGGGAGGTTTTTTAAAG(SEQ ID NO：9).

the probe for the target nucleic acid 2 (referred to as probe 2) includes: first oligonucleotide

SV40R1F3FAPh ATCAGCCATACCACATTTGTAGAGGTTTTAC(SEQ ID NO：10)，

SV40R1F3Dab CAAATGTGGTATGGCTGAT (SEQ ID NO: 4) with its 3' end linked to DABCYL.

The first and second oligonucleotides described above are combined in various ratios, typically 1: 2 to 1: 4, to form a partially double-stranded linear DNA probe. The formation of the first and second oligonucleotide hybrids brings the quencher and the fluorescent group into close proximity when the target nucleic acid is absent, effectively quenching the fluorescent signal. When the target nucleic acid is present, the first oligonucleotide preferentially hybridizes to the target sequence. As a result, the quencher is separated from the fluorescent group, resulting in an enhancement of fluorescence emission.

The first oligonucleotide in probes 1 and 2 is modified to contain a blocked 3' terminus so that it cannot be extended. However, when the first oligonucleotide binds to the target sequence, it is degraded by the 5' nuclease activity of a polymerase. Degradation of the first oligonucleotide of the probe (the consumed probe) results in a reduction in the amount of first oligonucleotide available for binding to the second oligonucleotide of the probe, so that an increase in the fluorescent signal can be measured at an appropriate temperature.

Melting profile analysis of probe 1, probe 2 and mixtures of probes 1 and 2 was performed using a Stratagene Mx3005 real-time PCR instrument (FIG. 20). The thermal parameters used were based on the parameters of the melting curve analysis software (fig. 20A): heating at 72 ℃ for 30 seconds, cooling to 40 ℃ and keeping for 30 seconds, then slowly heating to 94 ℃, and continuously collecting fluorescence emission data during the temperature rising period. Plotting the first negative derivative of the fluorescence emission readings with respect to temperature against temperature yields a curve, each peak of which corresponds to the actual T for that probe_m. One peak of probe 1 at 67 ℃ is its T_m(ii) a One peak of the probe 2 at 59 ℃ is its T_m(FIG. 20B).

Estimates of the percentage of double-stranded and single-stranded forms of each probe are listed in the table below. The actual calculation can also be performed by computer software.

The above estimation (calculation) can be made based on a multivariate view of the melting curve (fluorescence R vs. temperature, fig. 20C). Assuming that the baseline represents 100% double-stranded, R is 9000. R at one temperature (100% single stranded) is 2450. R was 13000 at 62 ℃. The difference in fluorescence values between 62 ℃ and baseline, dR 13000 and 9000 4000. The percentage of the double-stranded form of the probe at 62 ℃ was estimated to be 25.8% by 4000/(2450-.

Multiplex real-time PCR and Standard Curve analysis

The primer-probe premix is prepared as follows: the primers and probes were mixed to a final concentration of 0.4. mu.M for the probe and 0.6. mu.M for the primer, resulting in a 2X primer-probe premix.

By mixing equal amounts of 2X primer-probe premix and 2X TaqManGene Expression Master Mix (Applied biosystems, cat. No. 4369514) was mixed to obtain a reaction mixture.

The template DNA comprising the mixture of target nucleic acid 1, target nucleic acid 2 and target nucleic acids 1 and 2 was subjected to gradient dilution as follows: 1,0.1,0.01,0.001,0.0001,0.00001,0.000001.

Single plex PCR is performed using DNA samples containing target nucleic acid 1(k10) or target nucleic acid 2(SV 40). Double PCR was performed using a DNA sample comprising a mixture of target nucleic acid 1(k10) and target nucleic acid 2(SV 40).

The thermal parameters are: 8 minutes and 30 seconds at 95 ℃; 94 ℃ for 10 seconds, 66 ℃ for 20 seconds, 63 ℃ for 20 seconds, 54 ℃ for 30 seconds, 52 ℃ for 20 seconds, 61 ℃ for 20 seconds, 62 ℃ for 20 seconds, 68 ℃ for 20 seconds, 40 cycles; the fluorescence measurements were recorded at reading steps of 66 ℃,63 ℃, 54 ℃, 52 ℃, 61 ℃, 62 ℃ and 68 ℃.

The fluorescence emission (dR) at 62 ℃ was chosen as FE 1; the fluorescence emission (dR) at 52 ℃ was chosen as FE 2. From the tables listed above and the formula for calculating the Actual Consumption (ACA) in the discussion of the invention and in the claims we derive:

at 62 ℃, FE1 ═ 0.75 ═ ACA1) +0.05 ═ ACA2 (1)

At 52 ℃, FE2 ═ 100% ACA1+ 100% ACA2 (2)

According to (1) and (2),

ACA1＝(FE1-0.05*FE2)/0.7

ACA2＝(0.75*FE2-FE1)/0.7

for a double reaction in which both target nucleic acids are present, the above-mentioned FE1 and FE2 were obtained as follows:

FE1：

FE2：

the calculated ACAs of the target nucleic acid 1 are listed in the following table:

ACA1＝(FE1-0.05*FE2)/0.7

the amplification plot of ACA1 is shown in fig. 21A. The standard curve is shown in fig. 21B.

The calculated ACAs of the target nucleic acid 2 are listed in the following table:

ACA2＝(0.75*FE2-FE1)/0.7

an amplification plot of ACA2 is shown in fig. 21C. The standard curve is shown in fig. 21D.

In the case where only the target nucleic acid 1 is present in the reaction, the amplification pattern of ACA1 is shown in FIG. 18E, and the amplification pattern of ACA2 is shown in FIG. 21G. The results showed that, since only the target nucleic acid 1 was present in the reaction, its ACA1 showed a normal amplification curve and a normal standard curve (FIG. 21F), while its ACA2 showed a background curve, which indicated no signal from the target nucleic acid 2.

In the case where only the target nucleic acid 2 is present in the reaction, the amplification pattern of ACA1 is shown in FIG. 21H, and the amplification pattern of ACA2 is shown in FIG. 21I. The results showed that, since only the target nucleic acid 2 was present in the reaction, its ACA2 showed a normal amplification curve and a normal standard curve (FIG. 21J), while its ACA1 showed a background curve, which indicated no signal of the target nucleic acid 1.

Example 5

Amplification primers for target nucleic acid 1 (K10):

K10F155 CTCTGCTGACTTCAAAACGAGAAGAG(SEQ ID NO：5)；

K10R14 CCTGAGGGTTAAATCTTCCCCATTGA(SEQ ID NO：21)

K10R266Famph GTTCAATTGGGTTTCACCGCGCTTAGTTACA(SEQ ID NO：7)，

Amplification primers for target nucleic acid 2(SV 40):

dsredendF2 GTAAGATCCACCGGATCTAGATAAC(SEQ ID NO：8)；

sv40testR GGGAGGTGTGGGAGGTTTTTTAAAG(SEQ ID NO：9).

SV40R1F3FAPh ATCAGCCATACCACATTTGTAGAGGTTTTAC(SEQ ID NO：10)，

Amplification primers for target nucleic acid 3(Jak 2):

JknewF8 GTGGAGACGAGAGTAAGTAAAACTACA(SEQ ID NO：14)；

JKnewR8 CTCCTGTTAAATTATAGTTTACACTGACA(SEQ ID NO：15)；

the probe for the target nucleic acid 3 (referred to as probe 3) includes: first oligonucleotide

JKR3FamPh AACAGATGCTCTGAGAAAGGCATTAGA(SEQ ID NO：16)，

JKR3FDabF CTCAGAGCATCTGTT (SEQ ID NO: 12) whose 3' end was linked to DABCYL.

Amplification primers for target nucleic acid 4 (Kras):

KR12GVF1B GTCACATTTTCATTATTTTTATTATAAGGCCTGC(SEQ ID NO：17)；

KR12GVR12As GATCATATTCGTCCACAAAATGATTC(SEQ ID NO：18).

the probe for the target nucleic acid 4 (referred to as probe 4) includes: first oligonucleotide

KR12GVFamPh GAATATAAACTTGTGGTAGTTGGAGCTGT

(SEQ IDNO：19)，

KR12GVFAmdab CACAAGTTTATATTC (SEQ ID NO: 20) which is linked 3' to DABCYL.

The first oligonucleotide in all of the various probes is modified to include a blocked 3' terminus so that it cannot be extended. However, when the first oligonucleotide binds to the target sequence, it is degraded by the 5' nuclease activity of a polymerase. Degradation of the first oligonucleotide of the probe (the consumed probe) results in a reduction in the amount of first oligonucleotide available for binding to the second oligonucleotide of the probe, so that an increase in the fluorescent signal can be measured at an appropriate temperature.

Multiplex real-time PCR and Standard Curve analysis

The thermal parameters are: 8 minutes and 30 seconds at 95 ℃; 94 ℃ for 10 seconds, 66 ℃ for 20 seconds, 63 ℃ for 20 seconds, 54 ℃ for 30 seconds, 49 ℃ for 20 seconds, 55 ℃ for 20 seconds, 61 ℃ for 20 seconds, 68 ℃ for 20 seconds, 40 cycles; the fluorescence measurements were recorded at reading steps of 66 ℃,63 ℃, 54 ℃, 49 ℃, 55 ℃, 61 ℃ and 68 ℃. Various combinations of target nucleic acids present in the reaction were used, and some results are shown in FIG. 22: reaction A has no template; reaction B contains a target 3 template; reaction C contains target 2 template; reaction D contains target 4 template; reaction E contains target 1 and target 3 templates; reaction F contained target 1, 3 and 4 templates.

Non-keyword character of SEQUENCE listing (SEQUENCE LISTING FREE TEXT)

SEQ ID Nos: 1, 2, 5, 7 and 21 <223> PCR primers derived from K10

SEQ ID Nos: 3,4, 6 and 8-10 <223> PCR primers derived from SV40

SEQ ID Nos: 11-16 <223> PCR primers derived from Jak2

SEQ ID Nos: 17-20 <223> Kras-derived PCR primers

Claims

1. A method for testing a sample for one or more target nucleic acids, the method comprising:

(i) one or more pairs of forward/reverse oligonucleotide primers, wherein said pair of primers is capable of amplifying one or more target nucleic acids if present in said sample,

(ii) two or more probes, wherein each probe comprises

A first oligonucleotide comprising a first region and a second region, the first region being complementary to a portion of a target nucleic acid, and

at least one second oligonucleotide comprising a region complementary to said second region of said first oligonucleotide such that said first and second oligonucleotides are capable of forming a double stranded portion,

wherein each probe comprises a detectable label or a combination of detectable labels capable of producing a variable signal characteristic of the presence or absence of the double stranded portion between the first and second oligonucleotides of that probe, and

wherein at least two of the probes comprise the same detectable label or different detectable labels having indistinguishable emission spectra, and wherein the melting characteristics of the double stranded portion between the first and second oligonucleotides of each of such probes are different;

(b) performing an amplification reaction on said sample/reaction mixture under amplification conditions, wherein, when a target nucleic acid is present, said first oligonucleotide of a probe complementary to a portion of the target nucleic acid hybridizes to said target nucleic acid and is thereby consumed, wherein said consumed oligonucleotide of the probe is no longer capable of participating in the formation of the double-stranded portion of said probe; and

(c) measuring the melting profile of the double stranded portion between the first and second oligonucleotides of the unconsumed probe in the reaction mixture at least once as a function of temperature by detecting one or more signals from the labels in the unconsumed probe,

wherein the melt map provides an indication of whether at least one target nucleic acid is present in the sample,

wherein a first probe of the at least two of the probes has a melting temperature T according to the double-stranded portion of the first probe_m1，

WhereinA second probe of the at least two of the probes has a melting temperature T according to the double-stranded part of the second probe_m2，

Wherein T is_m1>T_m2，

Wherein the same label is independently attached to the first and second probes,

wherein at T_m1 and/or T_m2 provides an indication of the depletion of the one or more first and/or second probes.

2. The method according to claim 1, wherein the melting profile is measured before the reaction/amplification takes place, and/or is measured after the reaction/amplification is completed, and/or is measured during the reaction/amplification of each cycle or selected cycles,

wherein the method additionally comprises a step (d),

(i) comparing at least two melting profiles obtained in (c), and/or

(ii) Subjecting the melt map obtained in step (c)

Compared with a previously obtained dissolution curve of the same probe, or

Compared with the melting patterns of the same probe obtained simultaneously in parallel in a control reaction, or

Compared with the theoretical melting map of the same probe,

wherein the change in the melt map provides an indication of whether at least one target nucleic acid is present in the sample/reaction mixture,

wherein the pre-amplification melt profile is measured in the same reaction vessel prior to the start of the reaction/amplification or in a separate reaction vessel in which no amplification occurs due to the reaction mixture lacking one or more components necessary for the reaction/amplification,

wherein in step (d) the post-amplification or in-amplification melt profile is compared with the pre-amplification melt profile of the duplex of the probe to determine whether a particular probe is consumed as an indication that the corresponding target is present in the sample.

3. The method according to any one of the preceding claims, wherein at least one detectable label is a fluorescent label, wherein step (b) further comprises a step (b1) of obtaining a Fluorescence Emission (FE) cycle by cycle at each Measurement Temperature (MT), wherein the Fluorescence Emission (FE) is a baseline corrected fluorescence (dR).

4. The method of claim 3, wherein when the amplification reaction mixture comprises "n" probes for multiplex detection of "n" target nucleic acids, wherein a first probe has a melting temperature T_m1, the second probe has a melting temperature T_m2, the third probe has a melting temperature T_m3, the nth probe has a melting temperature T_mn, wherein T_ml>T_m2>T_m3...>T_mn, the percentage of double stranded form of each probe at a specific or different temperature is determined experimentally or is theoretically calculated by a computer program, wherein the first fluorescence emission FEa is obtained at a measurement temperature MTa at which more than 50% of the first probe is in duplex form and the second fluorescence emission FEb is obtained at a measurement temperature MTb at which more than 50% of the second probe is in duplex form and the n-1 th fluorescence emission FE (n-1) is obtained at a measurement temperature MT (n-1) at which more than 50% of the (n-1) th probe is in duplex form and the n-th fluorescence emission FEn is obtained at a measurement temperature MTn at which more than 80% of the n-th probe is in duplex form, and optionally, fluorescence emission FE0 is obtained at a measurement temperature MT0 at which MT0 no more than 10% of the first probe is in duplex form, where n is a positive integer and n.gtoreq.2.

5. The method of claim 3, wherein said step (b) further comprises a step (b2) of determining the actual consumption of fluorescence emission (ACA) for each probe cycle by cycle, wherein the kth probeActual consumption of fluorescence emission by ACA_kDescribed, wherein said kth probe has a ds (double stranded) form of a percentage of (dska)% at a specific measurement temperature (MTa) at which said fluorescence emission FEa contributed by said first probe will be (ds1 a)% (ACA)₁) The contribution from the second probe will be (ds2 a)% (ACA)₂) The contribution from said kth probe will be (dska)% (ACA)_k) The contribution from said nth probe will be (dsna)% (ACA)_n) Wherein the Actual Consumption (ACA) is calculated using the following formula:

at the measurement temperature MTa, the total fluorescence emission will be

FEa＝(ACA1)*(ds1a)％+(ACA2)*(ds2a)％+(ACA3)*(ds3a)％...+(ACAn)*(dsna)％

At the measurement temperature MTb, the total fluorescence emission will be

FEb＝(ACA1)*(ds1b)％+(ACA2)*(ds2b)％+(ACA3)*(ds3b)％...+(ACAn)*(dsna)％

At the measurement temperature MTc, the total fluorescence emission will be

FEc＝(ACA1)*(ds1c)％+(ACA2)*(ds2c)％+(ACA3)*(ds3c)％...+(ACAn)*(dsna)％

The rest are analogized, where the ACA for each probe can be calculated from the above formula, where ". DELTA.denotes" multiplied "," k "is a positive integer, 1. ltoreq. k.ltoreq.n, and" n "is the number of probes.

6. The method of claim 5, wherein the actual consumption of fluorescent emission by each probe is obtained by a computer program that calculates at each measured temperature at each cycle.

7. The method of claim 1, wherein the amplification is an isothermal amplification or a thermal cycling amplification reaction comprising two or more denaturation, annealing, and primer extension steps.

8. A method for testing a sample for one or more target nucleic acids, the method comprising:

two or more probes, wherein each probe comprises

wherein at least two of the probes comprise the same detectable label or different detectable labels having indistinguishable emission spectra, and wherein the melt characteristics of the double stranded portion between the first and second oligonucleotides of each of such probes are different and the melt characteristics are distinguishable in a melt map analysis;

(b) performing the reaction on the sample/reaction mixture, wherein the reaction is a primer extension reaction under extension conditions, wherein, when the target nucleic acid is present, the first oligonucleotide corresponding to the probe of the extendable primer hybridizes to the target nucleic acid and is thus consumed during the primer extension reaction, wherein the consumed oligonucleotide of the probe is no longer capable of participating in the formation of the double stranded portion of the probe; and

(c) measuring a melting profile of the double stranded portion between the first and second oligonucleotides of the unconsumed probe in the reaction mixture at least once as a function of temperature by detecting one or more signals from the label in the unconsumed probe,

wherein the melt map provides an indication of whether at least one target nucleic acid is present in the sample.

9. A method for testing a sample for one or more target nucleic acids, the method comprising:

two or more probes, wherein each probe comprises

(b) performing said hybridization reaction on said sample/reaction mixture under hybridization conditions, wherein, when a target nucleic acid is present, a first oligonucleotide of a probe complementary to a portion of the target nucleic acid hybridizes to the target nucleic acid and is thus consumed during said reaction, wherein said consumed oligonucleotide of the probe is no longer capable of participating in the formation of the double-stranded portion of said probe; and

10. A method for monitoring PCR amplification of at least two target nucleic acids, the method comprising:

(ii) two or more probes, wherein each probe comprises

wherein each probe comprises a fluorescent label or a fluorescent label/quencher pair capable of generating a variable signal indicative of the presence or absence of a double stranded portion between the first and second oligonucleotides of that probe, and

(b) performing an amplification reaction comprising thermal cycling of the sample/amplification reaction mixture

Wherein, when a target nucleic acid is present, the first oligonucleotide of the probe that is complementary to a portion of the target nucleic acid is consumed in the amplification reaction; and is

Wherein said step (b) further comprises the step (b1) of obtaining Fluorescence Emission (FE) cycle by cycle at each Measured Temperature (MT), wherein said Fluorescence Emission (FE) is baseline corrected fluorescence (dR),

wherein when the amplification reaction mixture comprises "n" probes for multiplex detection of "n" target nucleic acids, wherein the first probe has a melting temperature T_m1, the second probe has a melting temperature T_m2, the third probe has a melting temperature T_m3, the nth probe has a melting temperature T_mn, wherein T_ml>T_m2>T_m3...>T_mn, wherein the percentage of double stranded form of each probe at a specific temperature or different temperatures is determined experimentally or is theoretically calculated by a computer program, wherein the first fluorescence emission FEa is obtained at a measurement temperature MTa at which more than 50% of the first probe is in duplex form and the second fluorescence emission FEb is obtained at a measurement temperature MTb at which more than 50% of the second probe is in duplex form and the n-1 th fluorescence emission FE (n-1) is obtained at a measurement temperature MT (n-1) at which more than 50% of the (n-1) th probe is in duplex form and the n-th fluorescence emission FEn is obtained at a measurement temperature MTn at which more than 80% of the n-th probe is in duplex form, and optionally fluorescence emission FE0 is obtained at a measurement temperature MT0 at which MT0 no more than 10% of the first probe is in duplex form, where n is a positive integer and n.gtoreq.2,

wherein said step (b) further comprises a step (b2) of determining cycle by cycle the actual consumption of fluorescence emission (ACA) for each probe, wherein said actual consumption of fluorescence emission of kth probe is by ACA_kDescribed, wherein the kth probe has a percentage at a particular measured temperature (MTa) of(dska)% of ds (double stranded) form, at which measurement temperature MTa said fluorescence emission FEa contributed by said kth probe will be (dska)% (ACA)_k) The contribution from said nth probe will be (dsna)% (ACA)_n) Wherein the Actual Consumption (ACA) is calculated using the following formula:

at the measurement temperature MTa, the total fluorescence emission will be

FEa＝(ACA1)*(ds1a)％+(ACA2)*(ds2a)％+(ACA3)*(ds3a)％...+(ACAn)*(dsna)％

At the measurement temperature MTb, the total fluorescence emission will be

FEb＝(ACA1)*(ds1b)％+(ACA2)*(ds2b)％+(ACA3)*(ds3b)％...+(ACAn)*(dsna)％

At the measurement temperature MTc, the total fluorescence emission will be

FEc＝(ACA1)*(ds1c)％+(ACA2)*(ds2c)％+(ACA3)*(ds3c)％...+(ACAn)*(dsna)％

The rest analogies, where the ACA for each probe can be calculated from the above formula, where ". DELTA.denotes" multiplied "," k "is a positive integer, 1. ltoreq. k.ltoreq.n, and" n "is the number of probes,

wherein said actual consumption of fluorescence emission of each probe is obtained by a computer program which calculates at each measured temperature at each cycle, and

wherein said actual consumption of fluorescent emission of each probe is correlated with the degree of amplification of said target nucleic acid to which said probe is bound.

11. The method of any one of claims 1 and 8-10, wherein said depletion of probe is achieved by hybridization of said first oligonucleotide of said probe to said target sequence, followed by integration of said first oligonucleotide of said probe into an amplification product, wherein said first oligonucleotide is either an extendable primer or one of said forward/reverse oligonucleotide primer pair when said first oligonucleotide of said probe can be integrated into said amplification product.

12. The method according to any one of claims 1 and 8-10, wherein said consumption of probe is achieved by hybridization of said first oligonucleotide of said probe to said target sequence, followed by degradation of first and/or second oligonucleotide of said probe, wherein when said first oligonucleotide of said probe is degraded during said reaction, said reaction mixture comprises a double-strand dependent nuclease activity.

13. The method of any one of claims 1 and 8-10, wherein the probe comprises a first label and a second label, wherein the first label is a fluorophore and the second label is a quencher, or wherein the first label is a quencher and the second label is a fluorophore.

14. The method of claim 13, wherein the first label is attached to the first oligonucleotide and the second label is attached to the second oligonucleotide such that the first and second labels are in close proximity when an internal duplex of the probe is formed.

15. The method of claim 13, wherein the label is on one oligonucleotide of the probe, said one oligonucleotide being either the first oligonucleotide or the second oligonucleotide.

16. The method of claim 15, wherein the first oligonucleotide of the probe does not comprise a label and the second oligonucleotide of the probe comprises at least one label, wherein the second oligonucleotide comprises a first label and a second label, the first label is attached at or near one end of the second oligonucleotide and the second label is attached at or near the other end of the second oligonucleotide, such that when the second oligonucleotide is not hybridized to the first oligonucleotide, the second oligonucleotide is in a random coil or stem-loop structure, the structure being such that the first and second labels are in close proximity.

17. The method of claim 16, wherein the first oligonucleotide of the probe does not comprise a label and the second oligonucleotide of the probe comprises two labels, wherein the second oligonucleotide comprises a first label and a second label, the first label is attached at or near one end of the second oligonucleotide and the second label is attached at or near the other end of the second oligonucleotide, such that when the second oligonucleotide is not hybridized to the first oligonucleotide, the second oligonucleotide is in a random coil or stem-loop configuration, the configuration being such that the first and second labels are in close proximity.

18. The method of claim 15, wherein the first oligonucleotide does not comprise a label and the second oligonucleotide comprises a label, wherein the label is capable of altering the emission of a signal relative to the emission of the second oligonucleotide in single stranded form when the second oligonucleotide is hybridized to the first oligonucleotide to form the double stranded portion of the probe.

19. The method of claim 15, wherein the first oligonucleotide of the probe does not comprise a label, and wherein the probe comprises two second oligonucleotides capable of hybridizing proximally to different sites of the second region of the first oligonucleotide, wherein one of the second oligonucleotides is attached to a first label and the other second oligonucleotide is attached to a second label such that when the two second oligonucleotides hybridize to the first oligonucleotide, the two labels are brought into close proximity and one label affects a signal from the other label.

20. The method of claim 13, wherein the first and second oligonucleotides of a probe are linked by a linking moiety comprising a nucleotide or a non-nucleotide chemical linking moiety, allowing the first and second oligonucleotides to form a stem-loop structure, wherein the first and second oligonucleotides are labeled with first and second labels, respectively, such that when the probe forms an internal stem-loop structure, the labels are brought into close proximity and one label affects the signal from the other label.

21. The method of any one of claims 1 and 8-10, wherein the first region of the first oligonucleotide does not overlap with a second region of the first oligonucleotide.

22. The method of any one of claims 1 and 8-10, wherein the first region of the first oligonucleotide overlaps with or is embedded in the second region of the first oligonucleotide, wherein the first oligonucleotide hybridizes to the target sequence at the T of the duplex_mT of said duplex hybridized to said second oligonucleotide as compared to said first oligonucleotide_mHigher such that the first oligonucleotide forms a stronger hybrid with the target than the first/second oligonucleotide duplex if present and therefore melts at a higher temperature.

23. The method of any one of claims 1 and 8-10, wherein the first oligonucleotide comprises a third region that is identical to a sequence of a primer used in the amplification reaction.

24. The method of any one of claims 1 and 8-10, wherein both the first and second oligonucleotides of a probe are capable of being consumed during amplification.

25. The method of any one of claims 1 and 8-10, wherein the first oligonucleotide is blocked at the 3 'end, and wherein the second oligonucleotide is blocked at the 3' end.

26. A method for testing a sample for one or more variant nucleotides on a target nucleic acid, the method comprising:

(a) contacting a sample comprising a target nucleic acid with a reaction mixture comprising:

(ii) at least one pair of probes, wherein a first probe of the pair of probes comprises a sequence complementary to a wild-type target nucleic acid sequence and a second probe of the pair of probes comprises a sequence complementary to a target nucleic acid sequence comprising a variant nucleotide, wherein each probe of the pair of probes comprises

wherein each probe of the pair of probes comprises the same second oligonucleotide,

wherein each probe comprises a detectable label or a combination of detectable labels capable of producing a variable signal indicative of the presence or absence of a double stranded portion between the first and second oligonucleotides of that probe, and

(b) performing an amplification reaction on the sample/reaction mixture, wherein, when a target nucleic acid is present, the first oligonucleotide of the probe complementary to a portion of the target nucleic acid is consumed during amplification, and

wherein the melt map provides an indication of whether at least one target nucleic acid has been amplified in the sample/amplification reaction mixture.

27. The method of claim 26, wherein the identical oligonucleotide in the pair of probes comprises a universal base or inosine corresponding to a nucleotide of the variation in the target nucleic acid sequence, wherein the universal base is a 3-nitropyrrole 2' -deoxynucleoside, a 5-nitroindole, a pyrimidine analog, or a purine analog.

28. The method of any one of claims 1, 8-10, and 26, wherein the plurality of first oligonucleotides of different probes hybridize to different sites of the same strand of the target sequence.

29. A kit for detection against one or more target nucleic acids, the kit comprising a probe comprising:

15-150 nucleotides of a first oligonucleotide comprising a first region and a second region, said first region being complementary to a portion of a target nucleic acid, and

at least one second oligonucleotide of 4-150 nucleotides comprising a region complementary to said second region of said first oligonucleotide such that said first and second oligonucleotides are capable of forming a double stranded portion,

wherein each probe comprises a detectable label or a combination of detectable labels capable of producing a variable signal indicative of the presence or absence of the double stranded portion between the first and second oligonucleotides of that probe,

and wherein

(a) The first oligonucleotide of the probe does not comprise a label, the second oligonucleotide comprises a first label and a second label, wherein the first label is attached at or near one end of the second oligonucleotide and the second label is attached at or near the other end of the second oligonucleotide, such that when the second oligonucleotide is not hybridized to the first oligonucleotide, the second oligonucleotide is randomly coiled or is in a stem-loop structure that brings the first and second labels into close proximity, and wherein when the second oligonucleotide is hybridized to the first oligonucleotide, the two labels are held away from each other; or

(b) The first oligonucleotide does not comprise a label and the second oligonucleotide comprises a label, wherein the label is capable of altering detectable signal emission of the label relative to the emission of the label when the second oligonucleotide is in single stranded form when the second oligonucleotide is hybridized to the first oligonucleotide to form the double stranded portion of the probe; or

(c) The first oligonucleotide of the probe does not comprise a label, the probe comprises two second oligonucleotides capable of hybridizing adjacently to different portions of the second region of the first oligonucleotide, wherein one of the second oligonucleotides is attached to a first label and the other second oligonucleotide is linked to a second label such that when the two second oligonucleotides hybridize to the first oligonucleotide, the two labels are brought into close proximity and one label affects the signal from the other label.

30. A kit for detection against one or more target nucleic acids, the kit comprising a probe mixture comprising two or more probes, wherein each probe comprises:

at least one second oligonucleotide of 4-150 nucleotides comprising a region complementary to said second region of said first oligonucleotide such that said first and second oligonucleotides are capable of forming a double-stranded portion, wherein each probe comprises a detectable label or a combination of detectable labels capable of producing a variable signal indicative of the presence or absence of the double-stranded portion between said first and second oligonucleotides of that probe,

and wherein

(a) The first label is attached to the second region of the first oligonucleotide and the second label is attached to a region of the second oligonucleotide that is complementary to the second region of the first oligonucleotide such that the first and second labels are brought into close proximity when an internal duplex of the probe is formed; or

(b) The first oligonucleotide of the probe does not comprise a label, the second oligonucleotide comprises a first label and a second label, wherein the first label is attached at or near one end of the second oligonucleotide and the second label is attached at or near the other end of the second oligonucleotide, such that when the second oligonucleotide is not hybridized to the first oligonucleotide, the second oligonucleotide is randomly coiled or is in a stem-loop structure that brings the first and second labels into close proximity, and wherein when the second oligonucleotide is hybridized to the first oligonucleotide, the two labels are held away from each other; or

(c) The first oligonucleotide does not comprise a label and the second oligonucleotide comprises a label, wherein the label is capable of altering detectable signal emission of the label relative to the emission of the label when the second oligonucleotide is in single stranded form when the second oligonucleotide is hybridized to the first oligonucleotide to form the double stranded portion of the probe; or

(d) The first oligonucleotide of the probe does not comprise a label, the probe comprising two second oligonucleotides capable of hybridizing adjacently to different sites of the second region of the first oligonucleotide, wherein one of the second oligonucleotides is attached to a first label and the other second oligonucleotide is attached to a second label such that when the two second oligonucleotides hybridize to the first oligonucleotide, the two labels are brought into close proximity and one label affects the signal from the other label; or

(e) The first and second oligonucleotides of a probe are joined by a linking moiety comprising a nucleotide or a non-nucleotide chemical linking moiety, allowing the first and second oligonucleotides to form a stem-loop structure, wherein the first and second oligonucleotides are labeled with first and second labels, respectively, such that when the probe forms an internal stem-loop structure, the labels are brought into close proximity and one label affects the signal from the other label;

and wherein at least two of the probes comprise the same detectable label or different detectable labels having indistinguishable emission spectra, and wherein the melting characteristics of the double stranded portion between the first and second oligonucleotides of each of such probes are different.

31. The kit of claim 29 or claim 30, wherein at least one label is a fluorescent label.

32. The kit of part (a) or (c) of claim 29 or part (a), (b), (d) or (e) of claim 30, wherein the probe comprises a first label and a second label.

33. The kit of claim 32, wherein the first label is a fluorophore and the second label is a quencher, or wherein the first label is a quencher and the second label is a fluorophore.

34. A probe as defined in any of parts (a) to (c) of claim 29, use of a probe mixture according to claim 30 in a method as defined in any of claims 1 to 28.