NZ719238B2 - Nucleic acid probe with single fluorophore label bound to internal cytosine for use in loop mediated isothermal amplification - Google Patents

Abstract

The present invention provides novel probes for use in LAMP detection methods. The probes contain a single fluorophore label bound to an internal cytosine residue of the probe. The probes are particularly useful in the detection of chlamydia and gonorrhea infections in a patient.

Description

NUCLEIC ACID PROBE WITH SINGLE FLUOROPHORE LABEL BOUND TO INTERNAL CYTOSINE FOR USE IN LOOP MEDIATED ISOTHERMAL AMPLIFICATION The t invention generally relates to a probe for the detection of a c acid, a method using said probe and a kit of parts. Preferably the probe of the invention is useful in a method for the detection of nucleic acids derived from dia trachomatis and/or Neisseria gonorrhoeae and may be used in the diagnosis of dia and/or Gonorrhoea infections.

Nucleic acid amplification is one of the most valuable tools in the life sciences field, including application-oriented fields such as clinical medicine, in which diagnosis of infectious diseases, genetic disorders and genetic traits is particularly benefited. In on to the widely used PCR-based detection (Saiki R.K., Scharf,S., Faloona,F., Mullis,K.B., Horn,G.T., Erlich,H.A. and Arnheim,N. (1985) Science, 230, 354), several amplification s have been invented. Examples include nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR) and loop-mediated isothermal amplification (LAMP). PCR uses heat denaturation of double-stranded DNA products to promote the next round of DNA synthesis. 3SR and NASBA eliminate heat denaturation by using a set of ription and reverse transcription reactions to amplify the target sequence.

These methods can amplify target nucleic acids to a similar magnitude, all with a detection limit of less than 10 copies and within an hour or so. They require either a precision instrument for amplification or an elaborate method for detection of the amplified products due to poor specificity of target sequence selection. Despite the simplicity and the obtainable magnitude of ication, the requirement for a high precision thermal cycler in PCR ts this powerful method from being widely used, such as in e clinics as a routine diagnostic tool. In contrast, LAMP is a method that can amplify a few copies of DNA to over 100 in less than an hour under isothermal ions and with r specificity.

As with other molecular-probe based technologies identified above, loop-mediated isothermal ication (LAMP) assays can be used to detect the presence of ic rganisms in a sample. However, the detection methods are based on direct visual detection, turbidity or via a non-specific DNA intercalating dye. Direct visual measurement is end point measurement and is unable to provide real time analysis. Turbidity and nonspecific intercalating dyes do provide real time analysis of amplification which occurs however this is non-specific i.e. all amplification is detected whether this is true positive amplification or false amplification due to mis-priming, cross specificity.

Summary of the invention In a first aspect, the invention relates to a method of detecting a target nucleic acid sequence in a sample comprising: amplifying a target nucleic acid in the sample by loop-mediated isothermal amplification; probing the amplified nucleic acid with a probe comprising; an oligonucleotide probe sequence complementary to a region of a target nucleic acid ce, wherein said oligonucleotide probe ce has only one fluorophore label and which label is bound to an internal cytosine base and wherein said oligonucleotide probe sequence does not have a 3’ end terminator wherein the ne base is substantially lly disposed along the oligonucleotide’s length except for the positions 1-3 at the 3’ end and the position 1 at the 5’ end, wherein the probe is elongated and becomes incorporated into a DNA product during the loop-mediated isothermal amplification; and detecting the presence of the target nucleic acid, wherein an increase in fluorescence of the probe indicates the presence of the target nucleic acid in the sample.

In a second aspect, the invention s to a method of diagnosing Chlamydia and/or hea infection in a patient, comprising providing a sample derived from the patient; carrying out a method according to the first aspect; and detecting the presence of a nucleic acid derived from Chlamydia trachomatis and/or Neisseria gonorrhoeae, n an increase in the fluorescence of the probe tes the ce of a Chlamydia trachomatis and/or Neisseria gonorrhoeae infection.

In a third aspect, the invention relates to a probe for isothermal nucleic acid amplification comprising an oligonucleotide probe sequence mentary to a region of a target nucleic acid sequence, wherein said ucleotide probe ce has only one fluorophore label and which label is bound to an al cytosine base and wherein said oligonucleotide probe sequence does not have a 3’ end terminator, wherein the cytosine base is substantially centrally disposed along the oligonucleotide’s length except for the positions 1-3 at the 3’ end and the position 1 at the 5’ end; wherein the probe comprises one or more of the following sequences: SEQ ID NO. 3: TAAGATAAC[C-FAM]CCGCACGTG SEQ ID NO. 5: GCGAACATA [C-ALEXA546] CAGCTATGATCAA or SEQ ID NO. 6: ATGTTCA [C-JOE] CATGGCGGAG.

In a fourth aspect, the invention relates to a kit when used for detecting a target nucleic acid according to the method according to the first aspect, comprising a probe as specified in a method according to the first aspect, or as described in the third aspect, loop-mediated isothermal amplification reagent , enzyme, dNTPs and one or more loop-mediated isothermal amplification primers.

Description In accordance with a first embodiment, described is a probe for isothermal nucleic acid amplification comprising an oligonucleotide probe sequence complementary to a region of a target nucleic acid sequence, wherein said oligonucleotide probe sequence has only one fluorophore ligand and which ligand is bound to an internal ne base and wherein said oligonucleotide probe sequence does not have a 3’ end terminator.

In a preferred embodiment to oligonucleotide probe sequence is a DNA sequence and the target nucleic acid sequence is a DNA sequence.

Preferably, fluorescence increases to indicate the presence of the target nucleic acid in a sample.

The cytosine base is preferably substantially centrally disposed along the oligonucleotide’s . There are particular benefits associated with labeling the probe internally at a cytosine base. The specificity of the DNA product amplified in an isothermal reaction may be confirmed using a melt curve analysis. However due to a large number of product variants generated in this reaction and a low resolution of melt curve analysis, using intercalating dyes like V13, it is very difficult to guish between specific and unspecific DNA products generated under rmal conditions. Commonly used probes such as TaqMan® probe are not compatible with LAMP technology due to the strand displacement activity of BST polymerase. The probe of the invention is elongated and becomes incorporated into a DNA t during isothermal amplification, which allows for performing a melt curve is on the generated product. In the probe of the ion, the fluororphore is ated to an internal cytosine complementary to guanine in the antisense strand. Guanine affects the excitation state of many phores resulting in a formation of unique melt curve signatures and allows distinguishing n specific and unspecific products ted under isothermal conditions.

The oligonucleotide does not contain a ddNTP at its 3’ end which enables incorporation of the labelled oligonucleotide into the amplicon. Thus, the 3’ end of the probe is not "blocked".

The fluorophore may comprise any one or more selected from the following: FAM, JOE, TET, HEX, TAMRA, ROX, ALEXA and ATTO.

The probe may comprise the following sequence: ’ Xn C* Xm 3’ (SEQ ID NO. 1) Where n is >1, m is >3, X is nucleotide base; and * is a fluorophore. Preferably, the nucleotide base is selected from A, T, C and G. Preferably, n is more than 1 to 20 or less, more preferably more than 1 to 10 or less. Preferably, m is more than 3 to 20 or less, more preferably more than 3 to 10 or less. It is contemplated that all combinations of lengths of probe covered by the possible number of nucleotides that n or m make take by the preceding ranges are disclosed.

Preferably, the probe may comprise a sequence selected from any one of the following sequences: SEQ ID NO. 3: TAAGATAAC[C-FAM]CCGCACGTG (CT PB1-FAM internal) SEQ ID NO. 5: GCGAACATA [C-ALEXA546] TGATCAA (GC porA7-joe loopF) or SEQ ID NO. 6: ATGTTCA [C-JOE] CATGGCGGAG (GC glnA7-ALEXA546 loopB ).

The fluorescence is preferably sed when the oligonucleotide is incorporated into the target c acid sequence which results in a change in the configuration of the ampliconprobe complex g to an tion of the fluorophore excitation state.

The cytosine bound to the fluorophore ligand is not disposed at or proximate to the 5’ or 3’ end. More preferably it is not disposed in the first 3 bases from either the 5’ or 3’ end.

Preferably the cytosine bound to the fluorophore is disposed at the middle base of the probe.

In accordance with a further embodiment, described is an isothermal nucleic acid ication probe as described hereinabove.

In accordance with a further embodiment, described is a ediated isothermal ication probe as described above.

Methods and compositions for determining at least one target nucleic acid in a mixture of nucleic acids generally employ a probe, a hybridizing reagent, and one or more phosphate orming enzymes associated with any required nucleotide triphosphates to form a nucleic acid chain.

These methods usually involve amplification, such as including the use of a promoter in conjunction with a RNA polymerase, a restriction site where only one strand is cleaved and is then displaced by extension with a DNA polymerase, or a circular hybridizing t, where concatenated repeats are produced. ion of the amplified nucleic acid may take many forms but preferably via a fluorophore.

In accordance with a further embodiment, described is a method of detecting a target nucleic acid in a sample comprising: a. ying a target nucleic acid in the sample to provide an amplified nucleic acid; b. probing the amplified nucleic acid with a probe as described hereinabove; and c. detecting the presence of a single or multiple target nucleic acids.

The target nucleic acid may be that from a micro-organism, fungi, yeast, virus, human, animal, plant etc. The target nucleic acid for LAMP is known to enable LAMP primers and appropriately specific probes to be synthesised. Thus, the presence or absence of said micro-organism, fungi, yeast, virus, human, animal or plant in a sample can be determined.

Preferably the target nucleic acid is from Chlamydia trachomatis or ria gonorrhoeae.

Preferably, fluorescence increases to indicate the presence of the target nucleic acid in a sample.

The process is isothermal, and allows for amplification in a single stage or sequential stages in a single vessel, where all of the ts are ible.

In a further embodiment, described is a method of diagnosing Chlamydia and/or Gonorrhea in a patient, comprising providing a sample derived from the patient; adding one or more probes of the present invention to the ; and detecting the presence of a nucleic acid derived from Chlamydia trachomatis and/or Neisseria hoeae wherein an increase in the fluorescence of the probe indicates the presence of a Chlamydia trachomatis and/or Neisseria gonorrhoeae infection.

The sample may be treated by routine methods to enable the probe to bind with any target nucleotide present in the sample. Such treatment may include centrifuging and lysing the sample to release any target nucleic from the infecting microorganism.

In one embodiment, a single type of probe specific for a nucleic acid from either dia trachomatis or Neisseria gonorrhoeae is used in the method such that either only Chlamydia trachomatis or only Neisseria gonorrhoeae is detected in the sample.

In a preferred embodiment, at least two different probes are added to the sample wherein a first probe is labelled with a first fluorescent label and is ic for g Chlamydia trachomatis nucleic acid and a second probe is ed with a different fluorescent label to the first probe and is specific for probing Neisseria gonorrhoeae nucleic acid. In this embodiment, it is possible to simultaneously detect a Chlamydia and a Gonorrhea infection in a single sample d from a patient.

In one embodiment of the method bed herein, the sample from the patient may be a blood sample, urine sample, serum sample or saliva sample.

In ance with a r embodiment, described is a kit comprising a probe as described hereinabove, LAMP reaction buffer containing a polymerase enzyme, dNTPS and LAMP primers for the target.

In one embodiment a positive and negative control may be included in the kit. The reagents may be presented as wet reagents or in lyophilised form.

The buffer used in the method or kit of the invention comprises dNTPs at a concentration of from , one or more salts at a concentration of from 2-20mM, Tris pH8.8 at a concentration of from 10- 100mM, ose at a concentration of from 10-100mM, BST polymerase at an amount of from 1U-12U and 1% 1,2 propanediol.

The term "comprising" as used in this ication and claims means "consisting at least in part of". When interpreting statements in this specification, and claims which include the term "comprising", it is to be understood that other features that are additional to the features prefaced by this term in each statement or claim may also be present. Related terms such as "comprise" and "comprised" are to be interpreted in similar manner.

Abbreviations CT – Chlamydia trachomatis GC - Neisseria gonorrhoeae GlnA7 - Glutamine synthetase PorA7 – porin protein A7 LAMP –loop mediated isothermal amplification PCR – polymerase chain reaction.

The present invention will now be described, by way of example only, with reference to the ing examples and figures.

LAMP reaction V13 based detection of the target CT and GT DNA by LAMP was performed using LAMP V6.21 reaction buffer developed by the ant. Probe based detection of the target DNA was performed in V6.21p (without V13). The LAMP primer concentrations were as follows: CT PB1 - 0.8µM FIP & BIP primer, 0.2µM F3 & B3 and 0.4µM Loop s, GC porA7 and GC glnA7 – 2 µM FIP & BIP primer, 0.25µM F3 & B3 and 0.5µM Loop primers. All probes were used at a final concentration of 0.625µM. LAMP reactions were run for 60mins at a constant temperature of 63C using 0 real-time PCR machine. Readouts of the fluorescent signal were obtained in SybrGreen/FAM, Joe or Cy3 channel as appropriate.

Probe sequences SEQ ID NO. 2: GTGCACGC[C-FAM]CCAATAGAAT SEQ ID NO. 3: AAC[C-FAM]CCGCACGTG (CT PB1-FAM internal) SEQ ID NO. 4: TCGAGCAA[C-FAM]CGCTGTGAC[ddC] (CT PB1-FAM terminal) SEQ ID NO. 5: GCGAACATA [C-ALEXA546] TGATCAA (GC porA7-joe loopF) SEQ ID NO. 6: ATGTTCA [C-JOE] CATGGCGGAG (GC glnA7-ALEXA546 loopB ) or SEQ ID NO. 7: CCA GGG TAT CTA ATC CTG TTT G [C-FAM].

Target Sequences The target DNA sequences used in the Examples are SEQ ID No. 8: Chlamydia trachomatis G/SotonG1 plasmid pSotonG1 te sequence nk: HE603235.1) 1 tttgcaactc ttggtggtag actttgcaac tcttggtggt agactttgca actcttggtg 61 gtagacttgg tcataatgga cttttgttaa aaaatttctt aaaatcttag agctccgatt 121 ttgaatagct aaga aaatgggctc gatggctttc cataaaagta gattgttctt 181 tggg gacgcgtcgg aaatttggtt atctacttta ctaa ctagaaaaaa 241 ttatgcgtct gggattaact tttc tttagagatt ctggatttat cggaaacctt 301 gataaaggct atttctcttg gcga atctttgttt aagt ctctagatgt 361 ttttaatgga aaagtcgttt cagaggcctc taaacaggct agagcggcat gctacatatc 421 tttcacaaag tttttgtata gattgaccaa gggatatatt aaacccgcta ttccattgaa 481 agattttgga aacactacat tttttaaaat ccgagacaaa atcaaaacag aatcgatttc 541 taagcaggaa tggacagttt tttttgaagc gctccggata gtgaattata gagactattt 601 aatcggtaaa ttgattgtac aagggatccg taagttagac gaaattttgt ctttgcgcac 661 agacgatcta ttttttgcat agat ttcctttcgc attaaaaaaa gacagaataa 721 agaaaccaaa attctaatca catttcctat cagcttaatg gaagagttgc aaaaatacac 781 gaga aatgggagag tatttgtttc taaaataggg attcctgtaa caacaagtca 841 ggttgcgcat aattttaggc agtt ccatagtgct atgaaaataa aaattactcc 901 cagagtactt cgtgcaagcg ctttgattca tttaaagcaa ataggattaa aagatgagga 961 aatcatgcgt atttcctgtc tctcatcgag acaaagtgtg tgttcttatt ggga 1021 agaggtaagt cctctagtac aaacacccac aatattgtga tataattaaa attatattca 1081 tattctgttg aaaa cacctttagg ctatattaga gccatcttct ttgaagcgtt 1141 tcga gaggatttat cgtacgcaaa tatcatcttt gcggttgcgt gtcccgtgac 1201 cttcattatg tcggagtctg agcaccctag gcgtttgtac tccgtcacag cggttgctcg 1261 aagcacgtgc ggggttatct taaaagggat tgcagcttgt agtcctgctt gagagaacgt 1321 gcgggcgatt tgccttaacc ccaccatttt tccggagcga gttacgaaga caaaacctct 1381 tcgttgaccg atgtactctt gtagaaagtg cataaacttc taag ttataataat 1441 cctcttttct gtctgacggt tcttaagctg ggagaaagaa atggtagctt gttggaaaca 1501 aatctgacta atctccaagc ttaagacttc agaggagcgt ttacctcctt ttgt 1561 ctgggcgatc aaccaatccc gggcgttgat tttttttagc tcttttagga aggatgctgt 1621 ttgcaaactg ttcatcgcat ccgtttttac cctg aaaa atgttcgact 1681 attttcttgt ttagaaggtt gcgctatagc gactattcct tgagtcatcc tgtttaggaa 1741 tcttgttaag gaaatatagc ttgctgctcg aacttgttta gtaccttcgg tccaagaagt 1801 cttggcagag gaaacttttt taatcgcatc taggattaga ttatgattta aaagggaaaa 1861 ctcttgcaga ttcatatcca aagacaatag accaatcttt tctaaagaca aaaaagatcc 1921 tcgatatgat ctacaagtat gtttgttgag tgatgcggtc caatgcataa taacttcgaa 1981 gaag cttttcatgc gtttccaata ggattcttgg cgaattttta aaacttcctg 2041 ataagacttt tcgctatatt ctaacgacat ttcttgctgc aaagataaaa tccctttacc 2101 catgaaatcc ctcgtgatat aacctatccg caaaatgtcc tgattagtga cagg 2161 ttgttaacag gatagcacgc tcggtatttt tttatataaa catgaaaact cgttccgaaa 2221 atcg agat tatg cgttgttagg taaagctctg atatttgaag 2281 actctactga gtatattctg aggcagcttg ctaattatga gtttaagtgt tcccatcata 2341 aaaacatatt catagtattt aaatacttaa atgg attacctata actgtagact 2401 cggcttggga agagcttttg cggcgtcgta tcaaagatat ggacaaatcg tatctcgggt 2461 taatgttgca tgatgcttta tcaaatgaca agcttagatc cgtttctcat acggttttcc 2521 tcgatgattt gagcgtgtgt agcgctgaag aaaatttgag catt ttccgctcgt 2581 ttaatgagta caatgaaaat ccattgcgta gatctccgtt tctattgctt gagcgtataa 2641 agggaaggct tgatagtgct atagcaaaga ctttttctat tcgcagcgct agaggccggt 2701 atga tatattctca cagtcagaaa ttggagtgct ggctcgtata agac 2761 gagcagcgtt ctctgagaat caaaattctt tctttgatgg cttcccaaca ggatacaagg 2821 atattgatga taaaggagtt atcttagcta attt cgtgattata gcagctaggc 2881 catctatagg gaaaacagct ttagctatag acatggcgat aaatcttgcg gttactcaac 2941 agcgtagagt tggtttccta tctctagaaa tgagcgcagg tcaaattgtt gagcggattg 3001 ttgctaattt aata tctggtgaaa aattacaaag aggggatctc tctaaagaag 3061 aattattccg agtggaagaa gctggagaaa cagttagaga atcacatttt tatatctgca 3121 gtgatagtca gtataagctt aatttaatcg cgaatcagat ccggttgctg agaaaagaag 3181 atcgagtaga cgtaatattt atcgattact tgcagttgat caactcatcg gttggagaaa 3241 atcgtcaaaa tgaaatagca gatatatcta gaaccttaag agcc tcagagctaa 3301 acattcctat agtttgttta tcccaactat ctagaaaagt tgaggataga gcaaataaag 3361 ttcccatgct ttcagatttg cgagacagcg taga cgca gatgtgattt 3421 tgtttatcaa taggaaggaa tcgtcttcta attgtgagat aactgttggg aaaaatagac 3481 atggatcggt tttctcttcg gtattacatt tcgatccaaa aattagtaaa gcta 3541 ttaaaaaagt atggtaaatt atagtaactg ccacttcatc aaaagtccta tccaccttga 3601 aaatcagaag tttggaagaa gacctggtca atctattaag atatctccca aattggctca 3661 aaatgggatg gtagaagtta taggtcttga ttttctttca tctcattacc tagc 3721 agctatccaa agattactga cgaa ttacaagggg aacacaaaag gggttgtttt 3781 atccagagaa tcaaatagtt ttcaatttga aggatggata ccaagaatcc gttttacaaa 3841 aactgaattc ttagaggctt atggagttaa gcggtataaa acatccagaa ataagtatga 3901 gtttagtgga aaagaagctg aaactgcttt agaagccttg taccatttag aacc 3961 aata gtggcaacta gaactcgatg gactaatgga acacaaatag tagaccgtta 4021 ccaaactctt tctccgatca ttaggattta atgg ttaa ctgacgaaga 4081 aaatatagat atagacttaa caccttttaa atct acacggaaac ataaaggatt 4141 cgttgtagag ccatgtccta taga tcaaatagaa tttg taatcaagcc 4201 tgcaaatgta taccaagaaa tgcg tttcccaaac gcatcaaagt atgcttacac 4261 atttatcgac tgggtgatta cagcagctgc gaga cgaaaattaa ctaaggataa 4321 ttcttggcca gaaaacttgt tattaaacgt taacgttaaa agtcttgcat atattttaag 4381 gatgaatcgg tacatctgta caaggaactg aatc gagttagcta tcgataaatg 4441 tatagaaatc gccattcagc ttggctggtt atctagaaga aaacgcattg aatttctgga 4501 taaa ctctctaaaa aagaaattct atatctaaat aaagagcgct ttgaagaaat 4561 aactaagaaa tctaaagaac aaatggaaca agaatctatt aattaatagc aggcttgaaa 4621 ctaaaaacct aatttattta aaaa taaaaaagag ttttaaaatg ggaaattctg 4681 gtttttattt gtataacact gaaaactgcg tctttgctga taatatcaaa gttgggcaaa 4741 tgacagagcc gctcaaggac cagcaaataa tccttgggac aaaatcaaca cctgtcgcag 4801 ccaaaatgac agcttctgat ggaatatctt taacagtctc caataattca tcaaccaatg 4861 ttac aattggtttg gatgcggaaa aagcttacca gcttattcta gaaaagttgg 4921 gaaatcaaat tcttgatgga attgctgata ctattgttga tagtacagtc caagatattt 4981 tagacaaaat cacaacagac ccttctctag tgaa agcttttaac aactttccaa 5041 tcactaataa atgc ttat tcactcccag taacattgaa actttattag 5101 gaggaactga aataggaaaa ttcacagtca cacccaaaag ctctgggagc atgttcttag 5161 tctcagcaga tattattgca tcaagaatgg aaggcggcgt tgttctagct ttggtacgag 5221 aaggtgattc taagccctgc gcgattagtt atggatactc atcaggcgtt cctaatttat 5281 gtagtctaag aaccagcatt actaatacag gattgactcc aacaacgtat tcattacgtg 5341 taggcggttt agaaagcggt gtggtatggg ttaatgccct tggc aatgatattt 5401 taggaataac aaatacttct tctt ttttggaagt aatacctcaa acaaacgctt 5461 aaacaatttt attt ttcttatagg ttttatattt agagaaaaca gttcgaatta 5521 cggggtttgt tatgcaaaat aaaagaaaag tgagggacga ttttattaaa attgttaaag 5581 atgtgaaaaa agatttcccc gaattagacc taaaaatacg agtaaacaag gaaaaagtaa 5641 ctttcttaaa ttctccctta gaactctacc ataaaagtgt ctcactaatt ctaggactgc 5701 ttcaacaaat agaaaactct ttaggattat tcccagactc tcctgttctt gaaaaattag 5761 aggataacag tttaaagcta aaaaaggctt tgattatgct gtct agaaaagaca 5821 tgttttccaa ggctgaatag tact ctaacgttgg agttgatttg cacaccttag 5881 ttttttgctc ttttaaggga ggaactggaa aaacaacact ttctctaaac gtgggatgca 5941 acttggccca atttttaggg aaaaaagtgt tacttgctga cctagacccg caatccaatt 6001 tatcttctgg attgggggct agaa ataaccaaaa aggcttgcac gacatagtat 6061 caaa cgatttaaaa tcaatcattt gcgaaacaaa aaaagatagt gtggacctaa 6121 ttcctgcatc atttttatcc gaacagttta gagaattgga tattcataga ggacctagta 6181 acaacttaaa gttatttctg aatgagtact cttt ttatgacatc tgcataatag 6241 acactccacc tagcctagga gggttaacga cttt tgttgcagga gacaaattaa 6301 ttgcttgttt aactccagaa cctttttcta ttctagggtt acaaaagata cgtgaattct 6361 taagttcggt cggaaaacct gaagaagaac acattcttgg aatagctttg tctttttggg 6421 atgatcgtaa ctcgactaac caaatgtata tagacattat cgagtctatt tacaaaaaca 6481 agcttttttc aatt cgtcgagata tcag ccgttctctt cttaaagaag 6541 attctgtagc taatgtctat ccaaattcta gggccgcaga agatattctg aagttaacgc 6601 atgaaatagc aaatattttg catatcgaat atgaacgaga tcag aggacaacgt 6661 gaacaaacta aaaaaagaag cggatgtctt ttttaaaaaa aatcaaactg ccgcttctct 6721 agattttaag aagacacttc cttccattga actattctca gcaactttga attctgagga 6781 gagt ttggatcgat tatttttatc agagtcccaa aactattcgg atgaagaatt 6841 ttatcaagaa gacatcctag cggtaaaact gcttactggt cagataaaat ccatacagaa 6901 gcaacacgta cttcttttag gagaaaaaat ctataatgct agaaaaatcc tgagtaagga 6961 tcacttctcc tcaacaactt tttcatcttg gatagagtta agaa ctaagtcttc 7021 tgcttacaat gctcttgcat attacgagct ttttataaac ctccccaacc aaactctaca 7081 aaaagagttt caatcgatcc cctataaatc cgcatatatt ttggccgcta gaaaaggcga 7141 tttaaaaacc aaggtcgatg tgatagggaa agtatgtgga aact catcggcgat 7201 aagggtgttg gatcaatttc ttccttcatc tagaaacaaa gacgttagag aaacgataga 7261 taagtctgat ttagagaaga atcgccaatt tttc gaga tacttcgcat 7321 catatgttcc ggagtttctt tgtcctccta taacgaaaat cttctacaac agctttttga 7381 actttttaag caaaagagct gatcctccgt cagctcatat atatatttat tatatatata 7441 tttatttagg gatttgattt tacgagagag a SEQ ID No. 9: ria gonorrhoeae partial porA gene for class 1 outer membrane protein, isolate GC3 (GenBank: HE681886.1) 1 ggcg gcgcgacccg ttggggcaat agggaatcct ttgtcggctt ggcaggcgaa 61 ttcggcacgc tgcgcgccgg ccgcgttgcg aatcagtttg acgatgccag ccaagccatt 121 gatccttggg acagcaacaa tgatgtggct tcgcaattgg gtattttcaa acgccacgac 181 gatatgccgg tttccgtacg ctacgactcc ccggactttt ccggtttcag cggcagcgtc 241 caattcgttc cggctcaaaa cagcaagtcc gcctatacgc cggctcattg gactactgtg 301 tataacacta acggtactac tactactttc gctg ttgtcggcaa gcccggatcg 361 gatgtgtatt atgccggtct gaattacaaa aatggcggtt ttgccgggaa ctatgccttt 421 aaatatgcga ccaa tgtcggacgt aatgcttttg tctt gctcggcagt 481 gggagtgatg aagccaaagg taccgatccc aacc atcaggtaca ccgcctgacg 541 ggcggctatg gggaaggcgg cttgaatctc gcgg ctcagttgga tttgtctgaa 601 gaca aaaccaaaaa gacc gaaattgccg ccactgcttc ctaccgcttc 661 ggtaatacag tcccgcgcat cagctatgcc catggtttcg actttgtcga acgcagtcag 721 aaacgcgaac ataccagcta tga SEQ ID No. 10: ria gonorrhoeae glutamine synthetase (glnA) gene, glnA-14 allele, l cds (GenBank: AF520262.1) 1 cccgctttgt gcgc ttcaccgata ccaaaggcaa gcagcaccac tttaccgtgc 61 ctgcgcgcat cgtgttggaa gaccccgaag agtggtttga aaacggaccg gcgtttgacg 121 gctcgtccat cggcggctgg aaaggcattg aggcttccga tatgcagctg cgtcccgatg 181 cgtccacagc cttcgtcgat cctttttatg atgatgttac cgtcgtcatt acctgcgacg 241 tcatcgaccc tgccgacggt cagggttacg accgcgaccc gcgctccatc gcacgccgcg 301 ccgaagccta tttgaaatct tccggtatcg gcgacaccgc ctatttcggc cccg 361 aattcttcgt cttcgacggc gtagaatttg aaaccgacat gcacaaaacc cgttacgaaa 421 tcacgtccga cgcg tgggcaagcg gcctgcatat ggacggtcaa aacaccggcc 481 accgccccgc cgtcaaaggc ggctacgcgc ccgtcgcgcc gattgactgc gatt 541 ccgc catggtgaac attttggaag gactcggcat cgaagtcgaa gtccaccaca 601 gcgaagtcgg taccggcagc caaatggaaa tcggcacccg tttcgccact ttggtcaaac 661 gcgccgacca aacccaagat atgaaatacg tcatccaaaa cgttgcccac aatttcggca 721 aaaccgccac ctttatgccc aaaccgatta tgggcgacaa cggt atgcacgtcc 781 accaatccat ttggaaagac ggtcaaaacc tgttcgcagg cgacggctat gccggtttgt 841 ccgataccgc gctctactac atcggcggca tcatcaaaca cgccaaagcc ctgaacgcga 901 ttaccaatcc caac tcctacaaac gcctcgtgcc gcactttgaa gcaccgacca 961 aattggccta ttccgccaaa aaccgttccg tccg tatcccgtct gtgaacagca 1021 gcaaggcgcg cgaa gcgcgtttcc ccgacccgac cgccaacccg tatttggcat 1081 ttgccgccct gctgatggcc ggtttggacg gcattcaaaa caaaatccat ccgggcgacc 1141 ctgccgataa aaacctgtac gacctgccgc cggaagaaga cgcgctcgtc gtct 1201 gcgcttcttt ggaagaagca cttgccgccc tcaaggtcga ccacgaattc ctgctgcgcg 1261 gcggcgtgtt cagcaaagac tggatcgaca gctacatcgc ctttaaagag gaagatgtcc 1321 tccg tatggcgccg cacccgctgg aatttg The primer sequences used in the LAMP reaction are as follows: CT plasmid F3 TCTACAAGAGTACATCGGTCA (SEQ ID No. 11) B3 GTTGTCTTCTCG (SEQ ID No. 12) FIP GCAGCTTGTAGTCCTGCTTGAGTCTTCGTAACTCGCTCC (SEQ ID No.

BIP TCGAGCAACCGCTGTGACCCTTCATTATGTCGGAGTCTG (SEQ ID No. 14) LF1 CGGGCGATTTGCCTTAAC (SEQ ID No. 15) TACAAACGCCTAGGGTGC (SEQ ID No. 16) GC porA7 F3 ACCAAAAACAGTACGACCGA (SEQ ID No. 17) B3 AAGTGCGCTTGGAAAAATCG (SEQ ID No. 18) FIPATGGGCATAGCTGATGCGCGAATTGCCGCCACTGCTTC (SEQ ID No. 19) BIP TCGACTTTGTCGAACGCAGTCAAATCGACACCGGCGATGA (SEQ ID No. 20) LoopF1 GCGAACATACCAGCTATGATCAA (SEQ ID No. 21) GC glnA7 F3 TCATATCTTGGGTTTGGTCG (SEQ ID No. 22) B3 CTGCATATGGACGGTCAAA (SEQ ID No. 23) CGAAGTCCACCACAGCGAATTTGACCAAAGTGGCGAA (SEQ ID No.

FiP 24) CTTCGATGCCGAGTCCTTCCGATTGACTGCGGTCAAGAT (SEQ ID BiP No. 25) LF CAAATGGAAATCGGCACCC (SEQ ID No. 26) LB ATGTTCACCATGGCGGAG (SEQ ID No. 27) Buffer The Applicant has developed a buffer system for use with the probes of the invention and is designated V6.21 (or V6.21p without V13 dye present) in the following Examples. The trations of the buffer components are after buffer reconstitution: V6.21 4-10mM dNTP’s, 10mM salt, 30mM Tris pH8.8, 30mM Trehalose, 1-8U Bst polymerase, Dye and 0.05% ediol.

V6.21p 4-10mM dNTP’s, 10mM salt, 30mM Tris pH8.8, 30mM Trehalose, 1-8U Bst polymerase, and 0.05% propanediol.

CT/GC detection in clinical samples by ime PCR was performed using APTIMA CT/GC multiplex (Gen-Probe) according to the manufacturer’s instructions.

Agarose Gel Electrophoresis DNA electrophoresis was ted in 1% agarose gel 1xTAE buffer at 100V. LAMP DNA products were vitalized with GelRed (Invitrogen) with transilluminator.

V6.21 and V6.21p buffer were developed by the ant. LAMP primers were obtained from Eurofins. Fluorophore-labelled oligonucleotides were purchased from Integrated DNA technologies. Tris buffer, agarose gel and PCR grade water were purchased from Sigma. CT and GC DNA standards were obtained from ATCC.

Figures Figure 1 is a schematic of DNA probe as described herein. The probe ts of an oligonucleotide with an internal cytosine conjugated with a d fluorophore. The probe may be complementary to the internal region of the amplicon flanked by Fip and Bip primers or it may be a modified LoopF or LoopB primer internally labeled with a fluorophore.

Example 1 Figures 2A to 2F shows amplification plots generated with the CT PB1 (Figure 2A and Figure 2D), GC glnA7 (Figure 2B and Figure 2E) and GC porA7 (Figure 2C and Figure 2F) primers in V6.21 buffer containing V13 (Figures 2A, 2B and 2C) or V6.21p buffer without V13 dye (Figures 2D, 2E and 2F). The target ces shown in SEQ ID NOs. 8 to 10 with CT PB1 internal probe conjugated with FAM, GC glnA7 loop probe conjugated with Joe and GC porA7 loop probe conjugated with Alexa546 respectively. All reactions were performed for 60mins at a constant temperature of 63C with ABI7500 machine.

Figures 3A and 3B are melt curve es of LAMP products generated with CT PB1 primers in the presence of CT PB1 internal probe ated with FAM. 100pg per reaction of ATTC CT DNA standard was used as a positive control. A – normalized reporter plot, B – derivative reporter plot. Melt curve plots were generated based on the readouts in FAM channel with ABI7500 e.

Example 3 Figures 4A and B are melt curve analyses of LAMP product generated with GC glnA7 s in the ce of GC glnA7 loop probe conjugated with JOE. 100pg per reaction of ATTC GC DNA standard was used as a positive control. Figure 4A shows a normalized reporter plot and Figure 4B shows a tive reporter plot. Melt curve plots were generated based on the readouts in JOE channel with ABI7500 machine.

Figures 5A and 5B are melt curve analyses of LAMP product ted with GC porA7 primers in the presence of GC porA7 loop probe conjugated with ALEXA546. 100pg per reaction of ATTC GC DNA rd was used as a positive control. Figure 5A shows a normalized reporter plot, Figure 4B shows a derivative reporter plot. Melt curve plots were generated based on the readouts in Cy3 channel with ABI7500 machine.

Example 5 Figures 6A to 6D show the results of a test to confirm the DNA product specificity with a probe described herein in loop mediated isothermal amplification. The late ication time of the false positives (more than 30mins after the lowest target DNA concentration detectable in the LAMP reaction (100fg GC DNA) indicates that the unspecific amplification may be a result of primer dimer formation. The standard melt curve analysis does not allow to distinguish between the specific and unspecific product in this LAMP reaction, but the unspecific product may be recognized with the probe of the invention. GC DNA was amplified with GC porA7 primers and visualized with V13 dye or GC porA7-ALEXA546 probe as appropriate.

Example 6 Figure 7 shows the amplification plots generated with CT PB1 primers in V6.21 buffer containing V13 or V6.21p buffer without V13 dye but in the presence of CT PB1 terminal probe (complementary to loop region) with an internal C conjugated with FAM and 3’ terminator (3’ddC). Despite a successful amplification of the target DNA confirmed by excitation of the V13 dye in the control reaction, CT PB1 probe with 3’ terminator did not generate a positive signal.

Example 7 Figures 8A and 8B shows the ication plots generated in V6.21p buffer containing ROX in the ce of CT PB1 s and CT PB1 terminal probe with an internal cytosine conjugated with FAM (Figure 8A), and universal primers and 3’UP probe with 3’ terminal cytosine conjugated with FAM (Figure 8B). The first line represents signals generated by ROX, and the second line corresponds to the signal generated in the FAM channel. Binding of the probe with an internally labeled C to the target DNA s in FAM excitation. Binding of the probe with a 3’ end C labeled to the target does not alter the FAM tion state.

Example 8 Figures 9A to 9C show the amplification plots generated with CT PB1 primers in V6.21p buffer without V13 in the presence of CT PB1 al probe with an internal C conjugated with FAM and a reference dye (ROX). Figure 9A show raw data, readouts from the FAM channel in the first line and from the ROX channel in a second line. Figure 9B shows amplification plots (generated in FAM channel) normalized to ROX. Figure 9C shows derivative reporter melt curve plots.

Example 9 Figures 10A to 10C show the tion of CT PB1-FAM probe specificity. Figure 10A shows amplification plots generated with CT PB1-FAM probe in the presence of CT DNA and CT primers. As a control, two sets of reactions were med where unspecific genes, GC glnA7 and GC porA7 were amplified with the corresponding LAMP primers in the presence of CT PB1-FAM probe. In V6.21p buffer the ication plots in the presence of CT PB1 probe in the FAM channel were generated only when CT DNA was present in the reaction and no signal was generated when unspecific genes (GC glnA7 and GC porA7) were ied. No signal was also generated when an ific probe was used in a reaction where CT DNA was amplified with CT primers. Figure 10C shows data obtained in an analogous experiment but conducted in V6.21 buffer containing an intercalating dye V31.

Figure 10C shows DNA products generated in the experiment described in Figure 10A.

Example 10 Figures 11A and 11B shows the validation of CT PB1-FAM probe against APTIMA CT assay. Fifty clinical samples confirmed to be positive (n=29) (Figure 11A) or negative (n=21) (Figure 11B) for CT were tested in V6.21p buffer with CT PB1-FAM probe. Out of 50 samples 24 tested negative (Figure 11A) and 26 tested positive (figure 11B) for CT with CT PB1-FAM probe. There was 86% agreement between the Aptima and CT PB-FAM tests.

Example 11 s 12A and 12B show the amplification plots generated in CT/GC multiplex with CT PB1-FAM + GC porA7-Alexa546 probes. CT and GC DNA was amplified in separate reactions or in conjugation in V6.21p buffer in the presence of CT PB1-FAM and GC porA7- Alexa546 . The readouts were taken in Cy3 (Figure 12A) and FAM (Figure 12B) channels. The experiment ed that two DNA targets may be amplified and detected in a simultaneous on with FAM and Alexa546 labeled probes and that there was no cross reactivity between CT PB1 and GC porA7 primers and probes.

Example 12 Table1 shows a comparison between V13 LAMP for CT and GC, CT/GC Aptima and CT/GC multiplex (CT M + GC porA7-Alexa546). DNA extracted from 136 clinical s was tested with CT/GC Aptima multiplex, CT PB1 and GC porA7 primers in V6.21 buffer containing V13 or in a multiplex reaction in v6.21p buffer in the presence of CT PB1 and GC porA7 primers and CT PB1-FAM and GC porA7-Alexa546 . In a control experiment the samples were also tested in a simplex reaction with GC glnA7-joe probe. The table shows the agreement scores between the tests.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the e of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

Certain statements that appear herein are r than what appears in the statements of the invention. These statements are provided in the sts of providing the reader with a better understanding of the invention and its practice. The reader is directed to the accompanying claim set which s the scope of the invention.

Claims (23)

1. A method of detecting a target nucleic acid sequence in a sample comprising: amplifying a target nucleic acid in the sample by loop-mediated isothermal amplification; g the amplified nucleic acid with a probe comprising; an oligonucleotide probe sequence complementary to a region of a target nucleic acid sequence, n said oligonucleotide probe sequence has only one fluorophore label and which label is bound to an internal cytosine base and wherein said oligonucleotide probe sequence does not have a 3’ end terminator n the cytosine base is substantially centrally disposed along the oligonucleotide’s length except for the positions 1-3 at the 3’ end and the position 1 at the 5’ end, wherein the probe is elongated and s incorporated into a DNA product during the loop-mediated isothermal amplification; and ing the presence of the target nucleic acid, wherein an se in fluorescence of the probe indicates the presence of the target nucleic acid in the sample.

2. The method of claim 1, wherein the oligonucleotide probe sequence is a DNA sequence and the target nucleic acid sequence is a DNA sequence.

3. The method as claimed in any one of the preceding claims, wherein the fluorophore comprises any one or more selected from the following: FAM, JOE, TET, HEX, TAMRA, ROX, ALEXA and ATTO.

4. The method as claimed in claim 3, wherein the fluorophore is FAM, Joe or Alexa546.

5. The method as d in any one of the preceding claims, wherein the oligonucleotide probe sequence comprises the following sequence: 5’ Xn C * Xm 3’ (SEQ ID NO. 1) wherein n is > 1, m>3, X is nucleotide base; and * is phore and wherein the nucleotide base is ed from A, T, C and G, n is more than 1 up to 20, and m is more than 3 up to 20.

6. The method of claim 5 wherein n is more than 1 up to 10.

7. The method of claim 5 or 6 wherein m is more than 3 up to 10.

8. The method as claimed in any one of the preceding , wherein the oligonucleotide probe sequence comprises one or more of the following sequences: SEQ ID NO. 3: TAAGATAAC[C-FAM]CCGCACGTG SEQ ID NO. 5: GCGAACATA [C-ALEXA546] CAGCTATGATCAA or SEQ ID NO. 6: ATGTTCA [C-JOE] CATGGCGGAG.

9. The method as claimed in any one of claims 1-7, wherein the target nucleic acid is from a micro-organism, fungi, yeast or virus.

10. The method as claimed in any preceding claim, wherein the target c acid is from Chlamydia trachomatis or ria gonorrhoeae.

11. A method of diagnosing Chlamydia and/or hea infection in a patient, comprising providing a sample derived from the patient; carrying out a method according to any one of claims 1-8; and detecting the presence of a nucleic acid derived fromChlamydia trachomatis and/or Neisseria gonorrhoeae, wherein an increase in the fluorescence of the probe indicates the presence of a Chlamydia trachomatis and/or ria gonorrhoeae infection.

12. The method of claim 11, wherein a single type of probe specific for a nucleic acid from either Chlamydia trachomatis or Neisseria gonorrhoeae is added to the .

13. The method of claim 11, wherein at least two different probes are added to the sample wherein a first probe is labelled with a first fluorescent label and is specific for probing Chlamydia trachomatis nucleic acid and a second probe is labelled with a different fluorescent label to the first probe and is specific for probing Neisseria gonorrhoeae nucleic acid.

14. The method of any one of the preceding claims, wherein the probes are provided in a buffer system comprising dNTPs at a concentration of from 1-10mM, one or more salts at a concentration of each salt of from 2-20mM, Tris pH8.8 at a concentration of from 10- 100mM, Trehalose at a concentration of from 10-100mM, BST polymerase at an amount of from 1U-12U and 0.01%-1% 1,2 ediol.

15. The method of claim 14, wherein the one or more salts are selected from the group consisting of KCl, (NH4)2SO4 and MgSO4.

16. A probe for isothermal nucleic acid amplification comprising an oligonucleotide probe ce complementary to a region of a target nucleic acid sequence, wherein said oligonucleotide probe sequence has only one fluorophore label and which label is bound to an al cytosine base and wherein said oligonucleotide probe sequence does not have a 3’ end terminator, wherein the ne base is substantially centrally ed along the oligonucleotide’s length except for the positions 1-3 at the 3’ end and the position 1 at the 5’ end; wherein the probe comprises one or more of the ing sequences: SEQ ID NO. 3: TAAGATAAC[C-FAM]CCGCACGTG SEQ ID NO. 5: GCGAACATA [C-ALEXA546] CAGCTATGATCAA or SEQ ID NO. 6: ATGTTCA [C-JOE] CATGGCGGAG.

17. A kit when used for detecting a target nucleic acid according to the method of any one of claims 1 to 15, comprising a probe as specified in a method of any one of claims 1 to 15 or as claimed in claim 16, loop-mediated isothermal amplification reagent buffer, enzyme, dNTPs and one or more loop-mediated isothermal amplification primers.

18. The kit as claimed in claim 17, further comprising a positive and negative control.

19. The kit of claim 18, wherein the reagent buffer comprises dNTPs at a concentration of from 1-10mM, one or more salts at a concentration of from 2-20mM, Tris pH8.8 at a concentration of from 10-100mM, Trehalose at a concentration of from 10-100mM, BST polymerase at an amount of from 1U-12U and 0.01%-1% 1,2 propanediol.

20. The kit of claim 19, wherein the one or more salts are selected from the group consisting of KCl, SO4 and MgSO4.

21. A method as claimed in any one of claims 1 to 15, substantially as herein described with reference to any example thereof, with or without nce to the figures.

22. A probe as d in claim 16, substantially as herein described with reference to any e f, with or without reference to the figures.

23. The kit as claimed in any one of claims 17 to 20, substantially as herein described with reference to any example thereof, with or without nce to the figures. WO 63498 PCT/GBZOl-l/053238 J}... 3.x .m0