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1. WO2016094607 - PROCÉDÉS ET COMPOSITIONS POUR LA DÉTECTION DE BACTÉRIES RÉSISTANT AUX ANTIBIOTIQUES

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]

WHAT IS CLAIMED IS:

1. A method for detecting one or more NDM1 , KPC, IMP, VIM and OXA resistance- encoding genes in a sample, comprising:

(a) contacting a sample suspected of containing at least one of the NDM1 , KPC, IMP, VIM and OXA resistance-encoding genes with at least one primer comprising a sequence having substantial identity to at least one sequence selected from SEQ ID NO: 2, 3, 8, 9, 10, 1 1, 12, 14, 15, 16, 18, 19, 21, 22, 34, 35, 36, 39, 40, 44, 45, 46, 47, 50, 51, 52, 53, 56, 57, 58, 59, 62, 63, 66, 67, 69, 70, 72, 73, 75, and 76 to produce a mixture;

(b) incubating the mixture of step (a) under conditions sufficient to amplify NDM1 , KPC, IMP, VIM and OXA nucleic acids, thereby generating amplified nucleic acids; and

(c) detecting the amplified nucleic acids.

2. The method of claim 1 wherein the amplified nucleic acids comprise at least a portion of at least one of the NDM1, KPC, IMP, VIM and OXA resistance- encoding genes.

3. The method of claim 1 , wherein the amplified nucleic acids comprise at least a portion of more than one of the NDM1, KPC, IMP, VIM and OXA resistance- encoding genes.

4. The method of claim 1, wherein the step of detecting the amplified NDM1, KPC, IMP, VIM and OXA nucleic acids comprises using a fluorescence-generating probe comprising a sequence having substantial identity to at least one sequence selected from SEQ ID NO: 1, 4, 5, 6, 7, 13, 17, 20, 26, 27, 37, 38, 41, 42, 43, 48, 49, 54, 55, 60, 61 , 64, 65, 68, 71, 74, 77, 78, 79, 80, 81, 82, and 83, wherein Ra is a fluorophore with emission wavelength between about 400 and 900 nm, Rb is a non- fluorescent quencher, and wherein substitution of Ra and Rb allows quenching of fluorescence when the probe is unhybridized.

5. The method of claim 4, wherein the fluorescence-generating probe further comprises a minor groove binder.

6. The method of claim 5, wherein the minor groove binder is attached to the 3' end of the fluorescence-generating probe.

7. The method of claim 5, wherein the minor groove binder is attached to the 5' end of the fluorescence-generating probe.

8. The method of claim 4, wherein the fluorescence-generating probe comprises sequences having substantial identity to more than one sequence selected from SEQ ID NO: 1, 4, 5, 6, 7, 13, 17, 20, 26, 27, 37, 38, 41, 42, 43, 48, 49, 54, 55, 60, 61, 64, 65, 68, 71, 74, 77, 78, 79, 80, 81, 82, and 83.

9. The method of claim 1, wherein the primer comprises a sequence that is complementary to more than one portion of the NDM1, KPC, IMP, VIM and OXA nucleic acids.

10. The method of claim 1, further comprising the step of amplifying a control nucleic acid having a sequence of SEQ ID NO: 33.

1 1. The method of claim 10, wherein the step of amplifying a control nucleic acid comprises using a fluorescence-generating probe having substantial identity to SEQ ID NO: 25, wherein Ra is a fluorophore with emission wavelength between about 400 and 900 nm, Rb is a non-fluorescent quencher, and wherein substitution of Ra and Rb allows quenching of fluorescence when the probe is unhybridized.

12. The method of claim 10, wherein the step of amplifying a control nucleic acid comprises using one or more primers having substantial identity to a sequence that is SEQ ID NO: 23 or 24.

13. The method of claim 1, wherein p is 1.

14. A method for detecting one or more NDMl, KPC, IMP, VIM and OXA resistance- encoding genes in a sample, comprising:

(a) contacting a sample suspected of containing at least one of the NDMl, KPC, IMP, VIM and OXA resistance-encoding genes with at least one primer having the formula:


wherein X is a 5' portion of the primer that is non-complementary to the NDMl, KPC, IMP, VIM and OXA resistance-encoding genes and Y is a 3' portion of the primer that is substantially complementary to at least a portion of the NDMl, KPC, IMP, VIM and OXA resistance-encoding genes, and p is 0 or 1, to produce a mixture;

(b) incubating the mixture of step (a) under conditions sufficient to amplify NDMl, KPC, IMP, VIM and OXA nucleic acids, thereby generating amplified nucleic acids; and

(c) detecting the amplified nucleic acids using a fluorescence- generating probe comprising a sequence having substantial identity to at least one sequence selected from SEQ ID NO: 1, 4, 5, 6, 7, 13, 17, 20, 26, 27, 37, 38, 41, 42, 43, 48, 49, 54, 55, 60, 61, 64, 65, 68, 71, 74, 77, 78, 79, 80, 81, 82, and 83, wherein Ra is a fluorophore with emission wavelength between about 400 and 900 nm, Rb is a non-fluorescent quencher, and wherein substitution of Ra and Rb allows quenching of fluorescence when the probe is unhybridized.

15. The method of claim 14, wherein the amplified nucleic acids comprise at least a portion of at least one of the NDMl, KPC, IMP, VIM and OXA resistance- encoding genes.

16. The method of claim 14, wherein the amplified nucleic acids comprise at least a portion of more than one of the NDMl, KPC, IMP, VIM and OXA resistance- encoding genes.

17. The method of claim 14, wherein the primer comprises a sequence having substantial identity to at least one sequence selected from SEQ ID NO: 2, 3, 8, 9, 10, 11, 12, 14, 15, 16, 18, 19, 21, 22, 34, 35, 36, 39, 40, 44, 45, 46, 47, 50, 51, 52, 53, 56, 57, 58, 59, 62, 63, 66, 67, 69, 70, 72, 73, 75, and 76.

18. The method of claim 14, wherein the primer comprises sequences having substantial identity to more than one sequence selected from SEQ ID NO: 2, 3, 8, 9, 10, 11, 12, 14, 15, 16, 18, 19, 21, 22, 34, 35, 36, 39, 40, 44, 45, 46, 47, 50, 51, 52, 53, 56, 57, 58, 59, 62, 63, 66, 67, 69, 70, 72, 73, 75, and 76.

19. The method of claim 14, wherein the fluorescence-generating probe further comprises a minor groove binder.

20. The method of claim 14, wherein the fluorescence-generating probe comprises sequences having substantial identity to more than one sequence selected from SEQ ID NO: 1, 4, 5, 6, 7, 13, 17, 20, 26, 27, 37, 38, 41, 42, 43, 48, 49, 54, 55, 60, 61, 64, 65, 68, 71, 74, 77, 78, 79, 80, 81, 82, and 83.

21. The method of claim 14, wherein X is [A-B]m and Y is [A-B]n, wherein A represents a sugar phosphate backbone, modified sugar phosphate backbone, locked nucleic acid backbone, or a variant thereof in any combination, B represents a nucleic acid base or a modified base, m and n are integers of from about 4 to about 30.

22. The method of claim 14, further comprising the step of amplifying a control nucleic acid having a sequence of SEQ ID NO: 33.

23. The method of claim 22, wherein the step of amplifying a control nucleic acid comprises using a fluorescence-generating probe having substantial identity to SEQ ID NO: 25, wherein Ra is a fluorophore with emission wavelength between about 400 and 900 nm, ¾ is a non-fluorescent quencher, and wherein substitution of Ra and ¾ allows quenching of fluorescence when the probe is unhybridized.

24. The method of claim 22, wherein the step of amplifying a control nucleic acid comprises using one or more primers having substantial identity to a sequence that is SEQ ID NO: 23 or 24.

25. The method of claim 14, wherein p is 1.

26. A method for simultaneously detecting NDMl, KPC, IMP, VIM and OXA nucleic acids in a sample, comprising:

(a) contacting a sample suspected of containing NDMl, KPC, IMP, VIM or OXA nucleic acids with:

(i) at least one forward flap primer comprising at least one of the following sequences:

aataaatcataaGCAGACTGGGCAGTCGG (SEQ ID NO:3), aataaatAGGCAACCAAACCACTACGTTATCT (SEQ ID NO:l 1), aataaatAGGCAGCCAAACTACTAGGTTATCT (SEQ ID NO: 12), aataaatcaCGAATGCGC AGCACCI * GGATAGA (SEQ ID NO: 16), aataaatcataaCGCCATCCCTGACGATCAAAC (SEQ ID NO: 19), and aataaatcatTCTTGCCATTCCTTTGCTACCG (SEQ ID NO:22),

wherein the lowercase nucleotide sequence is non-complementary to sequences of the NDMl, KPC, IMP, VIM or OXA nucleic acids; and

(ii) at least one reverse flap primer comprising at least one of the following sequences:

aataaatcatGTCATTTGCCGTGCCATAC (SEQ ID NO:2), aataaatcatGGAATA*GAGTGGCTTAATTCTC (SEQ ID NO:8), aataaatcatGGAATA*GGGTGGCTTAATTCTC (SEQ ID NO:9), aataaatcatGGAATA*GAATGGCTTAACTCTC (SEQ ID NO: 10), aataaCGCATTCTCTAGAAGGACTCTCATC (SEQ ID NO: 14), aataaCGCACTCTCTAAAAGCGCTCTCCTC (SEQ ID NO: 15),

aataaatcataaGTCTGGCAGCACACTTCCTA (SEQ ID NO: 18), and aataaatcaATGCGTGTATTAGCCTTATCGGC (SEQ ID NO:21),

wherein the lowercase nucleotide sequence is non-complementary to sequences of the NDMl, KPC, IMP, VIM or OXA nucleic acids, to produce a reaction mixture;

(b) incubating the reaction mixture of step (a) under conditions sufficient to amplify the NDMl, KPC, IMP, VIM or OXA nucleic acids, thereby generating amplified nucleic acids; and

(c) detecting the amplified nucleic acids.

The method of claim 26, wherein the step of detecting the amplified nucleic acids comprises using a fluorescence-generating probe comprising at least one of the following sequences:

Rai-G*CAGGTTCCGGTTTTG-Rb, (SEQ ID NO:l)

R^-G* GI * C AC ACTCC AGATAAC-Rbi (SEQ ID NO:4)

Ra2-G*GI*CACACTCAAGATAAC-Rbi (SEQ ID NO:5)

Ra2- G*CTGA*A*TTAA*CI*AATGAGC-Rbi (SEQ ID NO:6)

Ra2- G*CTGA*A*TTAA*CI*AATGAAC- Rbl (SEQ ID NO: 7)

Ra2-G*TGCGCTTCGGTCC- RM (SEQ ID NO: 13)

Ra2-G*ACATGCCGGGTTTC- Rw (SEQ ID NO: 17)

Ra3-G*TGTTTTTGGTGGCATCG- Rbi (SEQ ID NO:20)

Ra5-G*GTGGCATCGATTATC-Rb2 (SEQ ID NO:26)

Ra6-G*TGTTTTTGGTGGCATCG-Rb3 (SEQ ID NO:27),

wherein Rai, R^, Ra3, Ra5, and Ra6 are fluorophores with emission wavelengths between about. 400 and 900 nm, Rb], Rb2, and Rb3 are non- fluorescent quenchers, and wherein substitution of Ra and Rb allows quenching of fluorescence when the probe is unhybridized.

28. The method of claim 26, further comprising the step of amplifying a control nucleic acid having a sequence of SEQ ID NO: 33.

29. The method of claim 28, wherein the step of amplifying a control nucleic acid comprises using a fluorescence-generating probe having substantial identity to SEQ ID NO: 25 wherein Ra is a fluorophore with emission wavelength between about 400 and 900 nm, Rb is a non-fluorescent quencher, and wherein substitution of Ra and Rb allows quenching of fluorescence when the probe is unhybridized.

30. The method of claim 28, wherein the step of amplifying a control nucleic acid comprises using one or more primers having substantial identity to a sequence that is SEQ ID NO: 23 or 24.

31. A kit for detecting NDM1, KPC, IMP, VIM and OXA resistance-encoding genes in a sample, comprising one or more primers having a sequence with substantial identity to a sequence selected from SEQ ID NO: 2, 3, 8, 9, 10, 11, 12, 14, 15, 16, 18, 19, 21, 22, 34, 35, 36, 39, 40, 44, 45, 46, 47, 50, 51, 52, 53, 56, 57, 58, 59, 62, 63, 66, 67, 69, 70, 72, 73, 75, and 76.

32. The kit of claim 31, further comprising one or more fluorescence-generating probes comprising a sequence having substantial identity to at least one sequence selected from SEQ ID NO: 1, 4, 5, 6, 7, 13, 17, 20, 26, 27, 37, 38, 41, 42, 43, 48, 49, 54, 55, 60, 61, 64, 65, 68, 71, 74, 77, 78, 79, 80, 81, 82, and 83, wherein Ra is a fluorophore with emission wavelength between about 400 and 900 nm, Rb is a non- fluorescent quencher, and wherein substitution of Ra and Rb allows quenching of fluorescence when the probe is unhybridized.

33. The kit of claim 31, further comprising one or more primers or probes for amplifying a control nucleic acid having sequences with substantial identity to a sequence that is SEQ ID NO: 23, 24, or 25, or combinations thereof.

34. A kit for detecting a CTX-M nucleic acid in a sample, comprising one or more fluorescence-generating probes comprising a sequence having substantial identity to at least one sequence selected from SEQ ID NO: 1, 4, 5, 6, 7, 13, 17, 20, 26, 27, 37, 38, 41, 42, 43, 48, 49, 54, 55, 60, 61, 64, 65, 68, 71, 74, 77, 78, 79, 80, 81, 82, and 83, wherein Ra is a fluorophore with emission wavelength between about 400 and 900 nm, Rb is a non-fluorescent quencher, and wherein substitution of Ra and Rb allows quenching of fluorescence when the probe is unhybridized.

35. The kit of claim 34, further comprising one or more primers comprising a sequence with substantial identity to a sequence selected from SEQ ID NO: 2, 3, 8, 9, 10, 11, 12, 14, 15, 16, 18, 19, 21, 22, 34, 35, 36, 39, 40, 44, 45, 46, 47, 50, 51, 52, 53, 56, 57, 58, 59, 62, 63, 66, 67, 69, 70, 72, 73, 75, and 76.

36. The kit of claim 34, further comprising one or more primers or probes for amplifying a control nucleic acid having sequences with substantial identity to a sequence that is SEQ ID NO: 23, 24, or 25, or combinations thereof.