WO2002088385A1 - Method of nucleic acid sequence detection using general primers and the related kits - Google Patents

Abstract

The invention disclosed a method of nucleic acid sequences detection and/or quantitative analysis using general primers. These general primers group include oligonucleotide primer pairs which can be specially linked to need checking sequences and activate the target nucleic acid amplification process, at least one of those primers is general primer. This general primer includes: (a) special combination areas, (b) general areas, these general areas lie in the 5' terminal of the general primer and they include reported molecule combination area and common primer area. The invention also provided the method of nucleic acid sequences detection using general primers, useful in the detection and/or quantitative analysis of gene expressing and pathogens of the samples easily, sensitively and exactly.

Description

 Universal template nucleic acid detection method and kit

 The present invention relates to the field of nucleic acid detection, and more particularly, to a universal template primer set and a method for detecting and / or quantifying a nucleic acid using the universal template primer set. Background technique

 Recently, in order to satisfy the rapid and accurate detection and / or quantification of pathogens (such as viruses, bacteria, fungi), and specific nucleic acid sequences in normal and abnormal genes, a large number of technologies have been developed. These techniques have broad applications in the detection and quantification of microorganisms in food, environmental samples, breeding animals, and other types of materials. In these cases, the presence of certain putative microorganisms needs to be monitored. Other applications include forensics, molecular pathology, anthropology, archeology, and biology.

 A common practice for such tasks is nucleic acid hybridization. This method is based on the ability of two nucleic acid strands to form a double-stranded structure under appropriate conditions, where the two nucleic acid strands contain complementary or substantially complementary sequences so that they can specifically bind. In order to detect and / or quantify a specific nucleic acid sequence (referred to as a "target sequence"), a labeled oligonucleotide ("probe") needs to be prepared that contains a sequence that is complementary to the target sequence. In order to sensitively detect and / or quantify trace amounts of genetic material, many more mature technologies have been developed that usually involve the amplification of a target nucleic acid (DNA or RNA) in a test sample and subsequent detection, 'including the polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), and automatic maintenance synthesis reaction (3SR).

 Although all of these techniques are useful tools for detecting and identifying trace amounts of target nucleic acids in samples, they have various problems that limit their applicability in routine operations in a clinical laboratory environment. One of the most difficult problems is that the conditions for amplifying the target nucleic acid in each test for subsequent detection and quantitative analysis are different. In other words, there are no uniform conditions conducive to test standardization.

 In addition, for current nucleic acid detection and / or quantification methods based on target sequence amplification, accurately detecting and / or quantifying pathogens with different genotypes or mutations, such as mutations that lead to drug resistance, has been a well-known challenge.

 Branched DNA (bDNA) is an emerging signal amplification technique that provides high repeatability and accuracy. However, the method itself has limited sensitivity and is difficult to be widely adopted by conventional laboratories.

 Therefore, there is an urgent need in the art to develop easy-to-use, sensitive, and accurate detection and / or quantification techniques for the analysis of pathogens and gene expression in samples. Summary of the Invention

The purpose of the present invention is to provide an easy-to-use, sensitive and accurate analysis technique that can be used to detect and / or quantify the expression of pathogens and genes in a sample. In a first aspect of the present invention, a method for detecting a nucleic acid is provided, which includes steps:

 (1) In a nucleic acid amplification reaction system containing a reporter molecule, a nucleic acid amplification reaction is performed on a sample to be tested using a universal template primer set, wherein the primer set includes a nucleic acid amplification reaction that specifically binds to a detected nucleic acid sequence and triggers a target nucleic acid amplification reaction. And at least one primer is a universal template primer, the universal template primer includes:

 (a) a specific binding region, which is located at the 3 'end of the universal template primer and is used to specifically bind to the detected nucleic acid sequence;

 (b) a universal region, which is located at the 5 'end of the universal template primer and has a reporter binding region and / or a common primer region, and the reporter can specifically bind to the reporter binding region or the complement of the reporter binding region And the state of the reporter molecule is different in the following two cases, thereby generating a detectable signal: (i) a reporter molecule binding region or a complementary strand of the reporter molecule binding region, and (ii) a reporter molecule Cut off or replaced

 (2) Detectable detectable signal generated by the reporter.

 In a preferred example, the number of the reporter molecules is greater than or equal to the number of universal template primers.

 In another preferred example, two or more different universal template primer sets are used, and these universal template primer sets respectively specifically bind to target sequences of different subjects, different target sequences of the same subject, and the same Different regions of the same target sequence of the subject, or a combination thereof, and the reporter binding regions of these universal template primer sets specifically bind to the same or different reporter molecules, respectively.

 In a second aspect of the present invention, a universal template primer set is provided, the primer set includes an oligonucleotide primer pair that specifically binds to a detected sequence and triggers a nucleic acid amplification reaction of interest, wherein at least one primer is a universal Template primer, the universal template primer includes:

 (a) a specific binding region, which is located at the 3 'end of the universal template primer and is used to specifically bind to the detected nucleic acid sequence;

 (b) a universal region, which is located at the 5 'end of the universal template primer and has a reporter binding region and / or a common primer region, and the reporter can specifically bind to the reporter binding region or the complement of the reporter binding region And the state of the reporter molecule is different in the following two cases, thereby generating a detectable signal: (i) a reporter molecule binding region or a complementary strand of the reporter molecule binding region, and (ii) a reporter molecule Cut off or replaced.

 In a preferred example, the universal template primer further has a spacer between the specific binding region and the reporter binding region, and / or a spacer between the common primer region and the reporter binding region, and the spacer is l -20bp, preferably 5-10bpo

 In another preferred example, the universal template primer set further includes an amplification template primer for increasing the number of detected nucleic acid templates.

In a third aspect of the present invention, a nucleic acid detection kit is provided, which contains the universal template primer set according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

 Figure 1 shows two structural schematic diagrams of the universal template primer of the present invention.

 Figure 2 shows various combinations of the universal template primer set of the present invention.

 Figure 3 shows several schematic diagrams of signal generation in the universal template detection of the present invention.

 FIG. 4 is a schematic diagram of a nucleic acid detection method of the present invention, wherein a universal template primer set used includes a universal template primer, and a reporter molecule thereof is bound to a reporter molecule binding region of the universal template.

 FIG. 5 is a schematic diagram of a nucleic acid detection method of the present invention, wherein a universal template primer set used includes a universal template primer, a conventional primer, a common primer, and an amplification template primer, and a reporter molecule thereof is combined with a universal template reporter molecule Binding zone. detailed description

 As used herein, the following words / terms have the following meanings, unless stated otherwise.

 "Nucleic acid": a ribonucleotide (RNA), a deoxyribonucleic acid (DNA), a polynucleotide analog of RNA or DNA, an oligonucleotide analog of RNA or DNA.

 "Template": The full-length or partial sequence of a nucleic acid molecule capable of being amplified by a nucleic acid polymerase. The template may be RNA or DNA, or an analogue thereof, and may be single-stranded, double-stranded, or partially double-stranded.

 "Universal Template (UT) Primer": A universal template primer is a synthetic oligonucleotide sequence. Referring to FIG. 1A, a 3 'end of a universal template primer has a specific binding region which is complementary to a target sequence and serves as a primer for extension in an amplification reaction. In addition, the universal template primer also contains a universal region, which is located 5 'to the universal template primer and has a reporter binding region (or its complementary sequence). The reporter can specifically bind to the reporter binding region or the complementary strand of the reporter binding region. Universal template primers can be synthesized by a variety of methods known to those skilled in the art.

 In addition, the 5 'end of the universal region can contain an additional common primer region, 0-25bp in length, for binding common primers. The length of the common primer region of a special form of universal template primer is 0bp, as shown in Figure 1B.

 As used herein, a "reporter binding region" refers to a region that binds to a reporter molecule on a universal template primer. Of course, the reporter can be directly bound to the reporter binding region on the universal template primer, or it can be bound to the complementary strand of the binding region (for convenience, the region of the universal template primer that is the same as the reporter sequence is still referred to as "Reporter Binding Region";).

In addition, a preferred universal template primer also has a gap between the specific binding region and the reporter binding region, and / or a gap between the common primer region and the reporter binding region, and the gap is 1-20 bp. (Preferably 3-10bp, more preferably 5-10bp). The main purpose of adding spacers is to: (1) adjust the Tm of universal template primers so that the Tm of each primer in the universal template primer set is close to or the same; (2) prevent certain secondary structures (such as hairpin structure, dimer, etc.) ); And / or (3) prevent steric hindrance. It should be understood, however, that spacers are not required in all cases, and sometimes do not. Depending on the sequence to be detected, those skilled in the art will be able to determine whether a universal template primer requires a spacer, and the spacer The length and composition of the district.

 A "reporter" is a molecule used to generate a detectable signal. The reporter is in a different state in the following two situations, thereby generating a detectable signal: (i) binding to the reporter binding region or its complementary sequence region, and (i i) the reporter is cut or replaced. Examples of suitable reporters are probes based on the principle of fluorescence resonance energy transfer (FRET) and rare-earth-containing oligonucleotide chains.

 Fluorescence resonance energy transfer (FRET): It is a signal detection principle that uses the interaction between the wavelengths of the excitation light and the emission light of different fluorescent substances to detect certain specific wavelengths of light. Probes that generate signals based on this principle are called fluorescence resonance energy transfer probes. Particularly suitable fluorescent resonance energy transfer probes include (but are not limited to): Taqman probes, molecular beacons (Figure 3D), FRET dual probes (Figure 2J), and peptide ide nucleic acid ) Signal Probe (PNA Signal Probe for short).

 A "Taqman probe" is an oligonucleotide with a reporter group at its 5 'end and a quencher group at its 3' end, said quencher group inhibiting the reporter group from being detectable Signal (such as fluorescence). Taqman probe design is prior art and relevant information can be obtained from publicly available sources (He id CA, Stevens J, Livak KJ, Will iams PM; Real Time Quantitaive PCR, Genome Res. 1996 Oc t .; 6 (10): 986-94).

 Figure 3 shows several schematic diagrams of signal generation in the universal template detection of the present invention. Figures 3A and 3B show the detectable signal generated by the Taqman probe due to cleavage, and Figure 3C shows the detectable signal generated by the FRET dual probe due to digestion or substitution. Figure 3D shows a detectable signal generated by molecular beacons due to hybridization.

 A "universal template" refers to a nucleic acid sequence, or an antisense nucleic acid sequence thereof, that is amplified by incorporating a universal template primer during the PCR process of the present invention. These universal templates have both a nucleotide sequence corresponding to a target sequence and a nucleotide sequence corresponding to a universal region of a universal template primer, including a reporter binding region, a common primer region, and a spacer region. These universal templates function as templates in subsequent PCR amplification cycles.

 The method disclosed in the present invention is in principle classified as a signal amplification method. The key is to use a suitable nucleic acid sequence containing artificial sequences as a template for signal amplification (herein referred to as "universal template"), so the corresponding method is called "universal template amplified nucleic acid analysis" (UT analysis) . UT analysis allows sensitive, accurate and standardized detection and / or quantification of target nucleic acid molecules.

 In the present invention, there is no limitation on the sample to be tested, as long as it contains genetic material. Representative test samples include (but are not limited to): DNA samples, RNA samples, and cDNA samples obtained from reverse transcription of RNA, as well as other forms of modified polynucleotides.

 In the universal template primer of the present invention, there is no particular limitation on the length of the specific binding region, and its length is usually 6-35bp, preferably 15-25bp. There is no particular limitation on the general-purpose area, and its length is usually 8-100 bp, preferably 20-60 bp, and more preferably about 30-50 bp.

Nucleotides contained in universal template primers are usually selected from A, T, C, and G. However, some other nucleotides are included in the universal template primer to produce special detection effects, such as increasing specificity, incorporating fluorescent molecules, or increasing binding to the template. Suitable examples are not limited to nucleotides selected from the group: isoG, isoC, 2'-0-methyl-G, 2'-0-methyl-C, and combinations thereof.

 In the reaction system of the present invention, the relationship between the number of reporter molecules and the universal template primer is not particularly limited. However, preferably, the number of reporter molecules should be greater than or equal to the number of universal template primers, and usually the ratio of reporter molecules to universal template primers is greater than 1: 1-10: 1, and more preferably 1.5: 1- 5: 1. Thus, in the reaction system, the universal template primers are basically in a state of binding to the reporter molecule.

 When the reporter is a Taqman probe, the Tm of the binding region between the Taqman probe and the universal template primer reporter should be higher than the Tm of the specific binding region of the universal template primer and the template. Usually, it is 2-15 higher, preferably 5- 12 ° C higher.

 In addition, one or more "amplification template primers" may be included in the universal template primer set of the present invention. As used herein, "amplification template primer" refers to a primer used to increase the number of nucleic acid sequences (templates) to be detected. Amplification template primers are bound upstream or downstream of the sequence to be detected, which can effectively increase the number of templates in the amplification reaction, thereby providing detection sensitivity. It should be understood that the "amplification template primer" is a conventional primer, but its role is to increase the number of templates to be detected.

 In the present invention, as shown in FIG. 2, the universal template primer set has various combinations, including (but not limited to):

 (1) The downstream is a universal template primer (and optionally a public primer bound to it), the upstream is a conventional primer, the reporter is combined with the universal template primer (Figure 2A), or the upstream and downstream primer positions are interchanged (Figure 2B);

 (2) The combination of the primer set of Figure 2A and an amplification template primer (Figure 2C), and the combination of the primer set of Figure 2B and an amplification template primer (Figure 2D). That is, the upstream and downstream are conventional primer pairs, the middle is a universal template primer, and the reporter is combined with the universal template primer.

 (3) A combination of two universal template primers and two amplification template primers (Figure 2E). That is, the upstream and downstream are conventional primer pairs, the middle is a pair of universal template primers facing each other, and the reporter is combined with the universal template primer.

 (4) The upstream and downstream are conventional primer pairs, the two general template primers in the middle are opposite, and the reporter is bound to the universal template primer (Figure 2F);

 (5) The downstream is a universal template primer, the upstream is a conventional primer, the reporter molecule is combined with the complementary sequence of the universal template primer (Figure 2G), or the upstream and downstream primer positions are interchanged (not shown);

 (6) The combination of the primer set of Figure 2G and an amplification template primer (Figure 2H), that is, the upstream and downstream are conventional primer pairs, the middle is a universal template primer, and the reporter is combined with the complementary sequence of the universal template primer or the universal template Primer orientation change;

 (7) The upstream is a conventional primer, and the downstream is a universal template primer. The reporter molecule is labeled on the universal template primer and the obituary molecule respectively (Figure 21), or the universal template primer orientation is changed (not shown); or the primer set of Figure 21 Combination with a primer for amplification template (Figure 2J);

 (8) The combination of Figure 2E + Figure 2G, that is, the upstream and downstream are conventional primer pairs, and the middle is a pair of reverse-facing universal template primers, and the reporter is combined with the complementary sequence of the universal template primer (not shown)

(9) The combination of Figure 2F + Figure 2G, that is, the upstream and downstream are conventional primer pairs, and the two general template primers in the middle To the contrary, the reporter is bound to the complementary sequence of the universal template primer (not shown);

 (10) The combination of Figure 2E + Figure 21, that is, the upstream and downstream are conventional primer pairs, the middle is a pair of opposite general template primers, and the reporter is labeled on the universal template primer and the reporter (not shown);

 (11) The combination of Figure 2F + Figure 21, that is, the upstream and downstream are conventional primer pairs, and the middle is two general-purpose template primers with opposite directions. The reporter is labeled on the universal template primer and the reporter (not shown).

 In the method of the present invention, one or more universal template primer sets can be placed on the same detected nucleic acid sequence at the same time. This allows simultaneous detection of different positions on the detected nucleic acid sequence.

 In the present invention, for a reporter molecule that is combined with a universal template primer, different universal template primers can bind the same reporter molecule, or different reporter molecules (for example, a reporter group with different fluorescence and corresponding bursts). (A reporter molecule that has an immobilized group, a reporter molecule that has the same or a different sequence).

 There is no particular limitation on the length of the universal template amplified by the universal template primer set. Expressed as the distance between the 3 'ends of the specific binding regions of the two primers of the universal template primer set on the template, usually lbp-10kb, preferably l-2kb, more preferably 1-500b, most preferably It is 1 to 100bp. Especially when the spacing is less than 100 bp, primer sets can be designed for, for example, highly conserved regions of pathogens, thereby reducing the false negative rate. In addition, the shorter the pitch, the more common the common template can be. The invention is further described below with reference to the drawings.

 Amplification mode (1). Nucleic acid amplification analysis using a primer set (combination shown in Figure 2A) containing a universal template primer and a conventional primer.

 See Figure 4 now. In this example, a universal template primer set is used, which includes a general template primer 1 and a conventional primer 2. In addition, the reaction system also includes a common primer 3 and a reporter molecule 4. In this example, the sample to be detected is RNA or DNA.

 Step 1: Place the primer set and the test sample in the reaction system.

 Step 2: Under appropriate conditions, annealing (or hybridization) occurs, that is, the specific binding region at the 3 'end of the universal template primer 1 binds to the target RNA or DNA sequence.

 Step 3: In reverse transcriptase (for RNA target sequence) or DNA polymerase (for DNA target sequence), the 3 'end of the template primer is extended to the 5' end of the target sequence to form an RNA / DNA or DNA / DNA double strand.

 Step 4: The resulting double-stranded nucleic acid is denatured under appropriate conditions to form a single-stranded target sequence and a newly synthesized DNA sequence (ie, a universal template). Or use RNase to hydrolyze the RNA strand to form a newly synthesized DNA sequence (ie, a universal template).

 If necessary, repeat steps 3-4 above under appropriate conditions.

 Step 5: Regular primer 2 binds to the complementary region of the newly synthesized DNA sequence.

 Step 6: In the presence of DNA polymerase, the 3 'end of the conventional primer 2 extends to the 5' end of the universal template sequence, and the double-stranded DNA (the newly synthesized DNA sequence in the double-strand is also the universal template). When the 3 'end of the conventional primer 2 is extended to the reporter molecule bound to the reporter binding region, the following three situations occur:

(1) the extension stops; (2) Continue to extend the report molecule from the universal template;

 (3) Continue to extend and cut off or destroy the reporter.

 In the first and second cases, the reporter is still intact and therefore does not produce a detectable signal. In the third case, since the quencher group on the 5 ′ end of the reporter (such as Taqman probe) is hydrolyzed by the 5 ′ nuclease activity of DNA polymerase (such as Taq polymerase), The reporter group at the 3 'end is no longer quenched, resulting in a detectable signal (such as a fluorescent signal).

 Step 7: In the next cycle, three conditions will occur during the annealing after denaturation:

 7a: Universal template primer 1 binds to a newly synthesized DNA sequence containing the sequence of conventional primer 2.

 7b: Common primer 3 binds to a newly synthesized DNA sequence containing the sequence of conventional primer 2.

 7c: Conventional primer 2 combined with a universal template containing the artificial primer 1 sequence (similar to step 5, not shown).

 Step 8: There are three cases:

 8a: The universal template primer 1 extends to the 5 'end of the DNA strand containing the sequence of the conventional primer 2 to form a double-stranded DNA. In the next cycle, these DNA double-strands are the new universal template.

 8b: The common primer 3 extends to the 5 'end of the DNA strand containing the sequence of the conventional primer 2 to form a double-stranded DNA. In the next cycle, these DNA double-strands are the new universal template.

 8c: As in step 6, when the 3 'end of the conventional primer 2 is extended to the reporter bound to the reporter binding region, the following 3 cases will occur −

(1) the extension stops;

 (2) Continue to extend the report molecule from the universal template;

 (3) Continue to extend and cut off or destroy the reporter.

In the first and second cases, the reporter is still intact and therefore does not produce a detectable signal. In the third case, since the quencher group on the 5 ′ end of the reporter (such as Taqman probe) is hydrolyzed by the 5 ′ nuclease activity of the DNA polymerase (such as Taq polymerase), the Taqman probe The reporter group at the 3 'end is no longer quenched, resulting in a detectable signal (such as a fluorescent signal).

 Step 9: Repeat steps 7 and 8.

 After several cycles, the principle of PCR is the same, and the detectable signal resulting from the reporter being cut off increases exponentially (or approximately exponentially). When the detectable signal is fluorescent, these signals can be measured in real time with a real-time fluorescence reader, such as Roche's LightCycler, or ABI GeneAmp 5700 or GeneAmp 7700; or a static fluorescence reader can be used after the nucleic acid amplification reaction is completed. Fluorescence measurement. Amplification mode (2), nucleic acid amplification analysis using the universal template primer set shown in Figure 2C

See Figure 5 now. In this example, a universal template primer 1, a conventional primer 2 and an amplification template primer 5 are used to form a primer set. The reaction system also includes a common primer 3 and a reporter 4. In this example, the sample to be detected is DNA or RNA. Step 1: Place the primer set and the test sample in the reaction system.

 Step 2: Under appropriate conditions, annealing (or hybridization) occurs, and two cases occur at this time:

 2a: The specific binding region at the 3 'end of the universal template primer 1 binds to the target RNA or DNA sequence.

 2b: Amplification template primer 5 binds to the target RNA or DNA sequence.

 Step 3:

 3a: Corresponding to 2a, under the action of reverse transcriptase (for RNA target sequences) or DNA polymerase (for DNA target sequences), the 3 'end of the universal template primer 1 extends to the 5' end of the target sequence to form RNA / DNA or DNA / DNA double strand.

 3b: Corresponding to 2b, under the action of reverse transcriptase (for RNA target sequence) or DNA polymerase (for DNA target sequence), the 3 'end of the template primer 5 is extended to the 5' end of the target sequence to form RNA / DNA. Or DNA / DNA double-stranded.

 Step 4: The resulting double-stranded nucleic acid is denatured under appropriate conditions to form a single-stranded target sequence and a newly synthesized DNA sequence (ie, a universal template). Or use ase to hydrolyze the RNA strand to form a newly synthesized DNA sequence (in the first case, a universal template is formed; in the second case, a target molecule nucleic acid sequence that is bound to conventional primer 2 is provided).

 If necessary, repeat steps 2, 3 and 4 (optional) under appropriate conditions.

 Step 5: In the next cycle, four conditions will occur during the annealing after denaturation:

 5a: Conventional primer 2 binds to a newly synthesized DNA strand containing universal template primer 1.

 5b: Conventional primer 2 binds to a newly synthesized DNA strand containing amplification template primer 5.

 5c: The specific binding region at the 3 'end of the universal template primer 1 binds to the target RNA or DNA sequence (same as step 2a, not shown).

 5d: Amplification template primer 5 binds to the target RNA or Li sequence (same as step 2b, not shown).

 Step 6:

 6a: Corresponding to 5a, in the presence of DNA polymerase, the 3 'end of the conventional primer 2 extends to the 5' end of the universal template sequence, and double-stranded DNA (the newly synthesized DNA sequence in this double-strand is also a universal template). When the 3 'end of the universal template primer 2 is extended to the reporter molecule bound to the reporter binding region, the following three situations occur:

(1) the extension stops;

 (2) Continue to extend the report molecule from the universal template;

 (3) Continue to extend and cut off or destroy the reporter.

 In the first and second cases, the reporter is still intact and therefore does not produce a detectable signal. In the third case, a detectable signal is generated.

 6b: Corresponding to 5b, in the presence of DNA polymerase, the 3 'end of the conventional primer 2 extends to the 5' end of the nascent strand containing the sequence of the amplification template primer 5 to form a double-stranded DNA. In the next cycle, these DNA double-strands are the new universal template.

6c and 6d: Corresponding to 5c and 5d, a new universal template is formed (same as steps 3a and 3b, not shown). Step 7: In the next cycle, during the annealing after denaturation, four things will happen: 7a _: Conventional primer 2 binds to a newly synthesized DNA strand containing universal template primer 1 (same as 5a, not shown). 7b: Conventional primer 2 binds to a newly synthesized DNA strand containing amplification template primer 5 (same as 5b, not shown). 7c: The specific binding region at the 3 ′ end of the universal template primer 1 binds to the target RNA or DNA sequence (similar to step 2a).

 7d: Amplification template primer 5 binds to the target RNA or DNA sequence (same as step 2b, not shown);

 7e: Common primer 3 binds to a newly synthesized DNA sequence containing the sequence of conventional primer 2.

 Step 8: During the next extension, the following situations may occur:

 8a. · In the presence of DNA polymerase, the 3 'end of conventional primer 2 extends to the 5' end of the universal template sequence, and double-stranded DNA (the newly synthesized DNA sequence in this double-strand is also a universal template). When the 3 'end of the universal template primer 2 is extended to the reporter molecule bound to the reporter binding region, the following three situations occur:

 (1) the extension stops;

 (2) Continue to extend the report molecule from the universal template;

 (3) Continue to extend and cut off or destroy the reporter.

 In the first and second cases, the reporter is still intact and therefore does not produce a detectable signal. In the third case, a detectable signal is generated.

 8b: Same as 6b, in the presence of DNA polymerase, the 3 'end of the conventional primer 2 extends to the 5' end of the nascent strand containing the sequence of the amplification template primer 5 to form a double-stranded DNA. In the next cycle, these double-stranded DNA are the new universal templates.

 8c: Same as 6c, forming a new universal template (similar to step 3a). "

 8d: Same as 6d to form a new universal template (similar to 3b).

 8e: The common primer 3 extends to the 5 'end of the DNA strand containing the sequence of the conventional primer 2 to form a double-stranded DNA. In the next cycle, these DNA double-strands are the new universal template.

 Step 9: Repeat steps 7 and 8.

 After several cycles, the principle of PCR is the same, and the detectable signal resulting from the reporter being cut off increases exponentially (or approximately exponentially). When the detectable signal is fluorescent, these signals can be measured in real time with a real-time fluorescence reader, such as Roche's LightCycler, or ABI GeneAmp 5700 or GeneAmp 7700; or a static fluorescence reader can be used after the nucleic acid amplification reaction is completed. Fluorescence measurement. In the present invention, the reporter can also bind to the complementary strand of the reporter binding region on the universal template (Figure 2G and Figure 2H), or use two universal templates and two conventional primers (a combination of Figures 2E and 2F), which The nucleic acid amplification process is basically the same as the amplification modes (a) and (b).

In the present invention, another way to generate a signal is to use a FRET dual probe, that is, a reporter signal group is labeled on a universal template and a reporter molecule, respectively. See Figures 3C and 3C '. At this time, the universal template primer set includes: a universal template primer 1 labeled with a FRET group such as FAM fluorescein, and a pair of conventional primers 2. In the reaction system, a reporter 4 (oligonucleotide) labeled with another FRET group such as Dabcyl is also included. Steps 1-5 are similar to steps 1-5 of the amplification mode (a).

 In step 6: corresponding to 5a, in the presence of DNA polymerase, the 3 'end of the conventional primer 2 extends to the 5' end of the universal template sequence, and the double-stranded DNA (the newly synthesized DNA sequence in the double-strand is also a universal template). When the 3 'end of the universal template primer 2 is extended to the reporter molecule bound to the reporter binding region, the following three situations occur:

 (1) the extension stops;

 (2) Continue to extend the report molecule from the universal template;

 (3) Continue to extend and cut off or destroy the reporter.

 In the first case, the reporter molecule binds to the universal template primer and quenches the FAM molecule on the universal template primer with the labeled Dabcyl, so no detectable signal is generated. In the second case, although the reporter is complete, because the nascent strand replaces the reporter, the mutual quenching between the reporter and the universal template primer is destroyed, resulting in a detectable signal (see Figure 3C '). In the third case, the quencher group of the reporter is hydrolyzed by the 5 'nuclease activity of the DNA polymerase (such as Taq polymerase), so the reporter on the universal template primer such as FAM is no longer quenched This results in a detectable signal (such as a fluorescent signal) (Figure 3C).

 Corresponding to 5b, in the presence of DNA polymerase, the 3 'end of the conventional primer 2 extends to the 5' end of the nascent strand containing the sequence of the amplification template primer 5 to form a universal template.

 Steps 7-9 are similar to steps 7-9 in the above amplification mode (1).

 After several cycles, the principle of PCR is the same, and the detectable signal due to the reporter being cut or replaced is also increased exponentially (or approximately exponentially). When the detectable signal is fluorescent, these signals can be measured in real time using a real-time fluorescence reader, such as Roche's LightCycler, or ABI GeneAmp 5700 or GeneAmp 7700; or a static fluorescence reader can be used after the nucleic acid amplification reaction is completed. Fluorescence measurement. The invention has obvious advantages over the prior art, and its main advantages include:

 (1) Easy to combine detection

 This universal template region can be incorporated and a sequence not included in the target sequence can be introduced into the newly synthesized DNA sequence. The newly synthesized DNA sequence can be used as a template for further DNA amplification. Regardless of the number of different target sequences, the newly synthesized DNA sequence is mostly the same-universal template region sequence, which allows different target sequences to be transformed into sequences with common characteristics. Therefore, the DNA sequence can continue to be processed or manipulated under the same or substantially the same conditions, and it is easy to implement a composite detection such as multiple tubes under the same conditions, or one tube with multiple target sequences.

 (2) Higher analytical sensitivity

 By using a primer set containing two universal template primers, or designing multiple universal template primer sets for different sites of a single target sequence, a higher level of signal-to-noise ratio in the single probe mode in the prior art can be generated.

In addition, the use of amplification template primers can further increase detection sensitivity. (3) High accuracy

 The universal template primer and universal template nucleic acid detection method of the present invention can reduce the false negative probability caused by the following factors:

 (a) the secondary structure at the probe (or primer) binding site of the target sequence, these secondary structures may effectively affect the binding of the probe (or primer);

 (b) Sequence changes at the probe (or primer) binding site of the target sequence. These changes can be caused by different subtypes or mutations. For example, in HCV, HCV is mutated or mutated due to the use of various drugs. Using conventional nucleic acid detection methods often results in false negatives. It is convenient to find highly conserved short regions (such as 100 bp or less) in the genetic material of various organisms (including pathogens). With the present invention, a universal template primer set can be designed for these short, highly conserved regions (such as 40-50bp), thereby reducing false negatives. According to the inventors' detection of HCV mutant strains using multiple pairs of universal template primer sets, it was found that the false negative rate was reduced by at least 50%.

 (c) The fragmentation of the long target sequence during sample processing or processing, which occurs in the middle region of the amplification of the long fragment, will cause the replication of the complementary A sequence to stop.

 (4) Simplify or eliminate multiple detection

 When multiple pathogens need to be detected, it is often necessary to perform a separate test for a certain pathogen. This is because the optimal reaction conditions for different detection reactions are different. Therefore, when performing nucleic acid amplification reactions in the same reaction tube, the efficiency of each amplification reaction is difficult to be close to the same, which makes it difficult to detect multiple pathogens in the same reaction tube at the same time. .

 With the technology of the present invention, because most of the universal templates are the same, that is, the universal template region, it is convenient to standardize the reaction conditions of PCR for detecting each pathogen, thereby realizing detection of multiple pathogens in one tube. This makes the technology of the present invention particularly suitable for applications such as blood donation sample testing.

 (5) Reduce the multiple analysis cost using fluorescent labeling technology

 In the prior art, for multiple target nucleic acid sequences to be detected, the same number of fluorescently labeled probes are required. In contrast, in the present invention, only one common reporting probe is required for detecting a plurality of target sequences, so the cost can be greatly reduced. The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. Example 1

 HCV virus detection with universal template primer nucleic acid amplification

 (R virus, universal template primer common primer region length Obp)

 In this embodiment, a universal template primer set including a universal template primer and a conventional primer is designed, and a part of the reaction process is shown in FIG. 4.

The PCR protocol is: reverse transcription from RNA to DNA: RT (50 ° C, 20 minutes, 95 ° C, 5 minutes); PGR: 50 ° C, 2 minutes, 94 ° C 5 minutes; 94 ° C 20 seconds; and 61 ° C 40 seconds, a total of 40 cycles. Universal template primers:

 5-AAGGAACAGG CGGCGACGAA TCAACGACAG AACGCAACCC AACGCTACTC- 3 '(SEQ ID NO:

1)

 Regular primers-

5'-GTGCCCCCGC AAGACT-3 '(SEQ ID NO: 2)

 The Taqman probe sequence used is:

 5'- TCGTCGCCGC CTGTTCCTTA-3 '(SEQ ID NO: 3)

 The reporter group is 6-carboxyfluorescein (6-FAM) (located at the 5 'end), and the quenching group is 6-carboxy-tetramethyl rhodamine, TAMRA) (located on the 3 'end).

 The detection instrument used in the test was ABI GeneAmp 5700, the excitation light source was a halogen lamp, and the wavelength was 488 nm.

 The above primers and corresponding Taqman probes were used for amplification in the ABI5700 Gene Amplifier. As a result, fluorescent samples with different dilutions showed positive fluorescence signals at different cycles (Ct), while negative samples and other non-specific control samples (samples containing other pathogens) appeared. No fluorescence signal appeared at the end of the amplification reaction (CtMO) . Example 2

 Universal template amplification detection for HBV virus

 (DNA virus, the length of the common primer region of the universal template primer is Obp)

 In this embodiment, a universal template primer set including a universal template primer and a conventional primer is designed, and a part of the reaction process is shown in FIG. 4.

 In the PCR. Protocol is: PCR: 50 ° C, 2 minutes, 94 ° C 5 minutes; 94 ° C 20 seconds; and 61 ° C 40 seconds, a total of 40 cycles.

 The universal template primers are:

 5-AAGGAACAGG CGGCGACGAA TCAACGACAG AACCAGCGAT AGCCAGGACA- 3 '(SEQ ID NO:

4)

 Regular primers:

 5'- CCTCCAATCA CTCACCAACC- 3 '(SEQ ID NO: 5)

 The Taqman probe sequence used is:

 5'-TCGTCGCCGC CTGTTCCTTA- 3 '(SEQ ID NO: 3)

 The reporter group, the quenching group, and the detection instrument used therein are the same as in Example 1.

The above primers and corresponding Taqman probes were used for amplification in the ABI5700 Gene Amplifier. As a result, positive signals with different dilutions showed fluorescence signals in different cycles (Ct), while negative samples and other non-specific control samples (samples containing other pathogens) showed no fluorescence at the end of the amplification reaction (Ct> 40) Letter number. Example 3

 Universal template amplification detection for HCV virus (RNA virus)

 In this embodiment, a universal template primer set including a universal template primer, a conventional primer, and a common primer is designed. The reaction process is shown in FIG. 4.

 The PCR protocol is: reverse transcription from RNA to DNA: RT (50 ° C, 20 minutes, 95 ° C, 5 minutes); PGR: 50 ° C, 2 minutes, 94 ° C 5 minutes; 94Ό20 seconds; and 61 ° C40 seconds, a total of 40 cycles.

 The universal template primers are:

 5- TCATCCACAT CCCACCTCAT CAGGAACAGG CGGCGACGAA TCAACGACAG AACGCAACCC AACGCTACTC-3 '(SEQ ID NO: 6)

 Regular primers:

 5'-GTGCCCCCGC AAGACT-3 '(SEQ ID NO: 2)

 The common primer sequence used is-5'- TCATCCACAT CCCACCTCAT-3 '(SEQ ID NO: 7)

 The Taqman probe sequence used is:

 5--TCGTCGCCGC CTGTTCCTTA- 3 '(SEQ ID NO: 3)

 The reporter group, the quenching group, and the detection instrument used therein are the same as in Example 1.

 The above primers and corresponding Taqman probes were used for amplification in the ABI5700 Gene Amplifier. As a result, fluorescence signals of different dilutions of positive samples appeared in different cycles (Ct), while negative samples and other non-specific control samples (samples containing other pathogens) showed no fluorescence at the end of the amplification reaction (Ct> 40). signal. Example 4

 Universal template amplification detection for HCV virus (RNA virus)

 In this embodiment, a universal template primer set including a universal template primer, a pair of conventional primers, and a common primer is designed. The reaction process is shown in FIG. 5.

 The PCR protocol is: reverse transcription from RNA to DNA: RT (50 ° C, 20 minutes, 95 ° C, 5 minutes); PCR: 50 ° C, 2 minutes, 94Ό 5 minutes; 94 ° C 20 seconds; and 61Ό40 seconds A total of 40 cycles.

 The universal template primers are:

 5- TCATCCACAT CCCACCTCAT CAGGAACAGG CGGCGACGAA TCAACGACAG AACGCAACCC AACGCTACTC-3 '(SEQ ID NO: 6)

 Regular primer 1:

 5'- GTGCCCCCGC AAGACT- 3 '(SEQ ID NO: 2)

 Regular primer 2:

5'- TGAGTGTCGTACAGC CTCCAGG-3 '(SEQ ID NO: 8) The common primer sequences used are:

 5'- TCATCCACAT CCCACCTCAT-3 '(SEQ ID NO: 7)

 The Taqman probe sequence used is:

 5'— TCGTCGCCGC CTGTTCCTTA-3 '(SEQ ID NO: 3)

 The reporter group, quenching group, and detection instrument used therein are the same as in Example 1.

 The above primers and corresponding Taqman probes were used for amplification in the ABI5700 Gene Amplifier. As a result, fluorescence signals of different dilutions of positive samples appeared in different cycles (Ct), while negative samples and other non-specific control samples (samples containing other pathogens) showed no fluorescence at the end of the amplification reaction (Ct> 40) signal. Example 5

 Universal template amplification detection for HCV virus (RNA virus) and HBV virus (DNA virus)

 In this embodiment, a universal template primer set including two universal template primers, two pairs of conventional primers, and a common primer is designed. The reaction process is shown in FIG. 5.

 The PCR protocol is: reverse transcription from RNA to DNA: RT (50 ° C, 20 minutes, 95 ° C, 5 minutes); PCR: 50 ° C, 2 minutes, 94 ° C 5 minutes; 94 ° C 20 seconds; And 61 Ό 40 seconds, a total of 40 cycles.

 The universal template primers for HCV virus are:

 5- TCATCCACAT CCCACCTCAT CAGGAACAGG CGGCGACGAA TCAACGACAG AACGCAACCC AACGCTACTC- 3 '(SEQ ID NO: 6)

 The universal template primers for HBV virus are:

 5'- TCATCCACAT CCCACCTCAT CAGGAACAGG CGGCGACGAA TCATCCAGTC TATGTTTCCC TCTTGTTGCT-3 '(SEQ ID NO: 9)

 HCV virus conventional primer 1:

 5'- GTGCCCCCGC AAGACT-3 '(SEQ ID NO: 2)

 HCV virus conventional primer 2: '

 5 *-TGAGTGTCGTACAGC CTCCAGG-3 '(SEQ ID NO: 8)

 HBV conventional primer 1:

 5'-CCTCCAATCA CTCACCAACC-3 * (SEQ ID NO: 5)

 HBV conventional primer 2:

 5'-AGTTTCCGTC CGAAGGTTT-3 '(SEQ ID NO: 10)

 The common primer sequences used are:

 5'— TCATCCACAT CCCACCTCAT-3 '(SEQ ID NO: 7)

 The Taqman probe sequence used is:

 5'-TCGTCGCCGC CTGTTCCTTA-3 '(SEQ ID NO: 3)

 The reporter group, the quenching group, and the detection instrument used therein are the same as in Example 1.

The above primers and corresponding Taqman probes were used for amplification in the ABI5700 Gene Amplifier. As a result, add HCV and / or HBV-positive samples with different dilutions show fluorescence signals in different cycles (Ct). Containing HBV and HCV-positive samples does not affect the detection sensitivity. The Ct value is mainly determined by the high concentration of virus. Negative samples and other non-specific control samples (samples containing other pathogens) showed no fluorescence signal at the end of the amplification reaction (Ct> 40). Example 6

 Universal template amplification for detection of HCV virus (RA virus).

 In this embodiment, a universal template primer set including a fluorescein-labeled universal template primer and a pair of conventional primers is designed. The reaction process is shown in Figure 8:

 The PCR protocol is: reverse transcription from RNA to DNA: RT (50 ° C, 20 minutes, 95 ° C, 5 minutes); PCR: 50 ° C, 2 minutes, 94 ° C 5 minutes; 94Ό 20 seconds; and 61 40 ° C for 40 cycles.

 The universal template primer sequence is −

5'-AAGGAACAGG CGGCGACGAA TCAACGACAG AACGCAACCC AACGCTACTC- 3 '(SEQ ID NO:

11)

 The reporting group used is 6-carboxyfluorescein (6-FAM) (located at the 5 'end)

 Regular primers-

5'-GTGCCCCCGC AAGACT- 3 '(SEQ ID NO: 2)

 Amplification template primers:

 5'- TGAGTGTCGTACAGC CTCCAGG- 3 '(SEQ ID NO: 8)

 The reporter probe sequence used is:-5'-TCGTCGCCGC CTGTTCCTTA- 3 '(SEQ ID NO: 3)

 The quenching group used therein is Dabcyl (ie, tetra (4-methylaminophenylazo) -benzoic acid (3 'end).

 The detection instrument used in the test was ABI GeneAmp 5700, the excitation light source was a halogen lamp, and the wavelength was 488 dishes.

 The above primers and corresponding Taqman probes were used for amplification in the ABI5700 Gene Amplifier. As a result, if only the conventional primer 1 and the universal template primer are used for amplification, the detection sensitivity may be 10 to 100 times different from that of the universal template primer set in which the conventional primer and the amplification template primer are added at the same time. When different dilutions were added, positive samples showed fluorescence signals at different cycles (Ct), while negative samples and other non-specific control samples (samples containing other pathogens) showed no fluorescence signals at the end of the amplification reaction (C 40). In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims appended to the present application.

Claims

Claim A universal template primer set, wherein the primer set includes:  An oligonucleotide primer pair that specifically binds to a detected nucleic acid sequence and triggers a target nucleic acid amplification reaction. At least one of the primers is a universal template primer. The universal template primer includes:  (a) a specific binding region, which is located at the 3 'end of the universal template primer and is used to specifically bind to the detected nucleic acid sequence;  (b) a universal region, which is located at the 5 'end of the universal template primer and has a reporter binding region and / or a common primer region, and the reporter can specifically bind to the reporter binding region or the complement of the reporter binding region And the state of the reporter molecule is different in the following two cases, thereby generating a detectable signal: (i) a reporter molecule binding region or a complementary strand of the reporter molecule binding region, and (ii) a reporter molecule Cut off or replaced.  The universal template primer set according to claim 1, wherein the reporter molecule comprises a fluorescence resonance energy transfer type signal probe and a rare-earth element-containing oligonucleotide chain.  The universal template primer set according to claim 1, wherein the specific binding region has a length of 6 to 35 bp, and the universal region has a length of 8 to 100 bp.  The universal template primer set according to claim 2, wherein the specific binding region is 15-25bp in length, the universal region is 20-50bp in length, and the fluorescence resonance energy transfer type The signal probe is selected from the group consisting of a Taqman probe, a FRET dual probe, a molecular beacon, and a PNA signal probe, and there is a spacer between the specific binding region and the reporter binding region, and / or a common primer region and There is a gap between the reporter binding regions, and the gap is 1-20 bp.  The universal template primer set according to claim 1, wherein the universal template primer contains a nucleotide selected from the group consisting of isoG, isoC, 2'-0-methyl-G, 2'- 0-methyl-C, and combinations thereof. .  The universal template primer set according to claim 1, further comprising an amplification template primer for increasing the number of nucleic acid templates to be detected.  7. A detection kit, characterized in that it contains the universal template primer set according to claim 1.  8. A method for detecting nucleic acid, characterized in that it includes steps:  α) In a nucleic acid amplification reaction system containing a reporter molecule, a universal template primer set is used to perform a nucleic acid amplification reaction with a test sample, wherein the primer set includes a specific binding to a detected nucleic acid sequence and triggers a target nucleic acid amplification reaction And at least one primer is a universal template primer, the universal template primer includes:  (a) a specific binding region, which is located at the 3 'end of the universal template primer and is used to specifically bind to the detected nucleic acid sequence; (b) a universal region, which is located at the 5 'end of the universal template primer and has a reporter binding region and / Or common primer region, the reporter molecule can specifically bind to the reporter molecule binding region or the complementary strand of the reporter molecule binding region, and the state of the reporter molecule is different in the following two cases, thereby generating a detectable signal: (I) binding to the reporter binding region or the complementary strand of the reporter binding region, and (ii) the reporter is cut or replaced;  (2) Detectable detectable signal generated by the reporter.  The method according to claim 8, wherein the test sample is selected from the group consisting of a DNA sample, an RNA sample, and a cDNA sample obtained by reverse transcription of RNA.  10. The method according to claim 8, wherein two or more different sets of universal template primers are used, and these universal template primer sets specifically bind to target sequences of different subjects, Different target sequences of the subject, different regions of the same target sequence of the same subject, or a combination thereof, and the reporter binding regions of these universal template primer sets specifically bind to the same or different reporter molecules, respectively.