Methods and compositions for detecting antibiotic resistant bacteria
Primers and probes specific to the genes encoding extended spectrum beta-lactamase that involves CTX-M groups 1 and 9 that cause extended beta-lactamase resistance in bacteria are described herein, with methods and kits for using these primers and probes to detect CTX-M groups 1 and 9 nucleic acids. In the methods described, nucleic acids present in a clinical or test sample obtained from a biological sample or tissue suspected of containing the CTX-M groups 1 and 9 gene are amplified and corresponding sequences for CTX-M groups 1 and 9 are detected. The amplified nucleic acid can be detected by a variety of state of the art methods, including fluorescence resonance energy transfer (FRET), radiolabels, enzyme labels, and the like.
1. A method for detecting a CTX-M nucleic acid in a sample, comprising:
(a) contacting a sample suspected of containing the CTX-M nucleic acid with at least one flap primer having the formula:
5′-[X] p -Y-3′ (I),
wherein X is a 5′ portion of the flap primer that is non-complementary to the CTX-M nucleic acid and Y is a 3′ portion of the flap primer that is substantially complementary to at least a portion of the CTX-M nucleic acid, X has a sequence that comprises AATAAATCATAA (SEQ ID NO: 51) or at least four consecutive bases of AATAAATCATAA (SEQ ID NO:
51), Y has a sequence that is substantially complementary to at least a portion of a sequence selected from the group consisting of SEQ ID NO: 1, 12, 19 or 29, and p is 0 or 1, to produce a mixture;
(b) incubating the mixture of step (a) under conditions sufficient to amplify the CTX-M nucleic acid, thereby generating an amplified CTX-M nucleic acid; and
(c) detecting the amplified CTX-M nucleic acid using a fluorescence-generating probe having a sequence with substantial identity to a sequence selected from the group consisting of SEQ ID NO: 4, 9, 10, 11, 18, 22, 26, 27, 28, 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, and 46, wherein G* is guanine analogue 6- amino-1H-pyrazolo [3 ,4-d]pyrimidin-4(5H)-one, R a is independently selected from (M) a -Fl and (M) a -Q, R b is independently selected from (M) a -Fl and (M) a -Q, M is a minor groove binder, a is 0 or 1,Fl is a fluorophore with emission wavelength between about 400 and 900nm, and Q is a non-fluorescent quencher, wherein substitution of R a and R b allows quenching of fluorescence when the probe is unhybridized, and wherein the CTX-M nucleic acid is a CTX-M nucleic acid of group 1, 9 or 15.
2. The method of claim 1 , wherein X and Y are each about 4 to about 30 nucleotides in length.
3. The method of claim 1 , wherein the fluorescence-generating probe further comprises a minor groove binder.
4. The method of claim 1 , wherein the flap primer has a sequence having substantial identity to a sequence selected from the group consisting of SEQ ID NO: 2, 3, 6, 7, 8, 13, 14, 16, 17, 20, 21, 24, 25, 30, 31, 33, and 34.
5. The method of claim 1 , wherein the flap primer comprises a sequence that is complementary to more than one portion of the CTX-M nucleic acid.
6. The method of claim 1 , 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 combination thereof, B represents a nucleic acid base or a modified base, m and n are integers of from about 4 to about 30.
7. The method of claim 1 , further comprising the step of amplifying a control nucleic acid.
8. The method of claim 7 , wherein the control nucleic acid has a sequence with substantial identity to SEQ ID NO: 47.
9. The method of claim 7 , wherein the step of amplifying a control nucleic acid comprises using a fluorescence-generating probe having substantial identity to SEQ ID NO: 50, wherein G* is guanine analogue 6-amino-1H-pyrazolo [3,4-d]pyrimidin-4(5H)-one, R a is independently selected from (M) a -Fl and (M) a -Q, R b is independently selected from (M) a -Fl and (M) a -Q, M is a minor groove binder, a is 0 or 1, Fl is a fluorophore with emission wavelength between about 400 and 900 nm, and Q is a non-fluorescent quencher, wherein substitution of R a and R b allows quenching of fluorescence when the probe is unhybridized.
10. The method of claim 7 , 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: 48 or 49.
11. A method for detecting a CTX-M nucleic acid in a sample, comprising:
(a) contacting a sample suspected of containing the CTX-M nucleic acid with at least one primer having a sequence with substantial identity to a sequence selected from the group consisting of SEQ ID NO: 2, 3, 6, 7, 8, 13, 14, 16, 17, 20, 21, 24, 25, 30, 31, 33, and 34, to produce a mixture;
(b) incubating the mixture of step (a) under conditions sufficient to amplify the CTX-M nucleic acid, thereby generating an amplified CTX-M nucleic acid; and
(c) detecting the amplified CTX-M nucleic acid, using a fluorescence-generating probe having a sequence with substantial identity to a sequence selected from the group consisting of SEQ ID NO: 4, 9, 10, 11, 18, 22, 26, 27, 28, 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, and 46, wherein G* is guanine analogue 6-amino-1 H-pyrazolo[3,4-d] pyrimidin-4(5H)-one, R a is independently selected from (M) a -FI and (M) a -Q, R b is independently selected from (M) a -FI and (M) a -Q, M is a minor groove binder, a is 0 or 1, Fl is a fluorophore with emission wavelength between about 400 and 900 nm, and Q is a non-fluorescent quencher, wherein substitution of R a and R b allows quenching of fluorescence when the probe is unhybridized, and wherein the CTX-M nucleic acid is a CTX-M nucleic acid of group 1, 9 or 15.
12. The method of claim 11 , further comprising the step of amplifying a control nucleic acid.
13. The method of claim 12 , wherein the control nucleic acid has a sequence of SEQ ID NO: 47.
14. The method of claim 12 , wherein the step of amplifying a control nucleic acid comprises using a fluorescence-generating probe having substantial identity to SEQ ID NO: 50, wherein G* is guanine analogue 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, R a is independently selected from (M) a -Fl and (M) a -Q, R b is independently selected from (M) a -Fl and (M) a -Q, M is a minor groove binder, a is 0 or 1, Fl is a fluorophore with emission wavelength between about 400 and 900 nm, and Q is a non-fluorescent quencher, wherein substitution of R a and R b allows quenching of fluorescence when the probe is unhybridized.
15. The method of claim 12 , 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: 48 or 49.
16. A method for detecting a CTX-M nucleic acid in a sample, comprising:
(a) contacting a sample suspected of containing the CTX-M nucleic acid with at least one flap primer having the formula:
5′-[X] p -Y-3′ (I),
wherein X is a 5′ portion of the flap primer that is non-complementary to the CTX-M nucleic acid and Y is a 3′ portion of the flap primer that is substantially complementary to at least a portion of the CTX-M nucleic acid, and p is 0 or 1, to produce a mixture;
(b) incubating the mixture of step (a) under conditions sufficient to amplify the CTX-M nucleic acid, thereby generating an amplified CTX-M nucleic acid; and
(c) detecting the amplified CTX-M nucleic acid using a fluorescence-generating probe having a sequence with substantial identity to a sequence selected from the group consisting of SEQ ID NO: 4, 9, 10, 11, 18, 22, 26, 27, 28, 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, and 46, wherein G* is guanine analogue 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, R a is independently selected from (M) a -Fl and (M) a -Q, R b is independently selected from (M) a -Fl and (M) a -Q, M is a minor groove binder, a is 0 or 1, Fl is a fluorophore with emission wavelength between about 400 and 900 nm, and Q is a non-fluorescent quencher, wherein substitution of R a and R b allows quenching of fluorescence when the probe is unhybridized, and wherein the CTX-M nucleic acid is a CTX-M nucleic acid of group 1, 9 or 15.
17. The method of claim 16 , wherein X has a sequence that comprises AATAAATCATAA (SEQ ID NO: 51) or at least four consecutive bases of AATAAATCATAA (SEQ ID NO: 51), and Y has a sequence that is substantially complementary to at least a portion of a sequence selected from the group consisting of SEQ ID NO: 1, 12, 19 or 29.
18. The method of claim 16 , wherein the flap primer has a sequence with substantial identity to a sequence selected from the group consisting of SEQ ID NO: 2, 3, 6, 7, 8, 13, 14, 16, 17, 20, 21, 24, 25, 30, 31, 33, and 34.
19. The method of claim 16 , further comprising the step of amplifying a control nucleic acid.
20. The method of claim 19 , wherein the control nucleic acid has a sequence of SEQ ID NO: 47.
21. The method of claim 19 , wherein the step of amplifying a control nucleic acid comprises using a fluorescence-generating probe having substantial identity to SEQ ID NO: 50, wherein G* is guanine analogue 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, Ra is independently selected from (M)a-Fl and (M)a-Q, Rb is independently selected from (M)a-Fl and (M)a-Q, M is a minor groove binder, a is 0 or 1, Fl is a fluorophore with emission wavelength between about 400 and 900 nm, and Q is a non-fluorescent quencher, wherein substitution of Ra and Rb allows quenching of fluorescence when the probe is unhybridized.
22. The method of claim 19 , 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: 48 or 49.
23. A method for simultaneously detecting CTX-M groups 1 and 9 nucleic acids in a sample, comprising:
(a) contacting a sample suspected of containing the CTX-M groups 1 and 9 nucleic acids with:
(i) at least one forward flap primer comprising
5′-AATAAATCATAATCGGGTCGCCGGGAATG-3′ (SEQ ID NO: 25), and
(ii) at least one reverse flap primer comprising at least one of the following sequences: and
5′-AATAAATCATGCGATGAGACGTTTCGTCTGGA-3′ (SEQ ID NO:24), to produce a reaction mixture;
(b) incubating the reaction mixture of step (a) under conditions sufficient to amplify the CTX-M groups 1 and 9 nucleic acids, thereby generating amplified CTX-M groups 1 and 9 nucleic acids; and
(c) detecting the amplified CTX-M groups 1 and 9 nucleic acids.
24. The method of claim 23 , wherein the step of detecting the amplified CTX-M groups 1 and 9 nucleic acids comprises using fluorescence-generating probes comprising at least one of the following sequences:
(SEQ ID NO: 22)
R a -G*TCGGCTCGGTACGG-R b ,
and
(SEQ ID NO: 26)
R a -G*TAGGTTCAGTGCGATCC-R b ,
wherein G* is guanine analogue 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, Ra is independently selected from (M)a-Fl and (M)a-Q, Rb is independently selected from (M)a-Fl and (M)a-Q, M is a minor groove binder, a is 0 or 1, Fl is a fluorophore with emission wavelength between about 400 and 900 nm, and Q is a non-fluorescent quencher, wherein substitution of Ra and Rb allows quenching of fluorescence when the probe is unhybridized.
25. The method of claim 23 , further comprising the step of amplifying a control nucleic acid having a sequence of SEQ ID NO: 47.
26. The method of claim 25 , wherein the step of amplifying a control nucleic acid comprises using a fluorescence-generating probe having substantial identity to SEQ ID NO: 50, wherein G* is guanine analogue 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, Ra is independently selected from (M)a-Fl and (M)a-Q, Rb is independently selected from (M)a-Fl and (M)a-Q, M is a minor groove binder, a is 0 or 1, Fl is a fluorophore with emission wavelength between about 400 and 900 nm, and Q is a non-fluorescent quencher, wherein substitution of Ra and Rb allows quenching of fluorescence when the probe is unhybridized.
27. The method of claim 25 , 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: 48 or 49.
28. The method of claim 24 , wherein the at least one forward flap primer further comprises 5′-AATAAATCATAAGCGGATCGCCCGGAAT-3′ (SEQ ID NO: 21) and the at least one flap reverse primer further comprises 5′-AATAAATCATAAACGAAACGTTCCGTCTCGAC-3′ (SEQ ID NO: 20).