METHODS, COMPOSITIONS AND KITS FOR DETECTION AND ANALYSIS OF ANTIBIOTIC-RESISTANT BACTERIA

• US 20090081663A1
• Filed: 04/18/2008
• Published: 03/26/2009
• Est. Priority Date: 04/19/2007
• Status: Active Grant

• Current Assignee: Molecular Detection Inc.
• Original Assignee: Molecular Detection Inc.
• Inventors: Paitan, Yosef
• Granted Patent: US 8,017,337 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The present invention relates generally to detection of antibiotic-resistant bacteria in a sample. In particular, the invention provides methods, compositions and kits for detecting and analyzing methicillin-resistant Staphylococcus aureus (MRSA) and other methicillin-resistant bacteria in a sample.

Claims

1. A method of detecting methicillin-resistant S. aureus (MRSA) in a sample, said method comprising:

(a) providing a first set of primers, wherein said first set of primers are complementary to at least a portion of a mecA polynucleotide sequence;

(b) providing a second set of primers, wherein said second set of primers are complementary to at least a portion of a bridging region;

(c) providing a third set of primers, wherein said third set of primers are complementary to at least a portion of an S. aureus-specific polynucleotide sequence, and wherein said S. aureus-specific polynucleotide sequence is not an orfX polynucleotide;

(d) combining said first, second and third set of primers with said sample in a reaction mixture;

(e) performing a multi-cycle amplification reaction with said reaction mixture;

(f) determining cycle numbers of appearance of each of said mecA, bridging region and S. aureus-specific polynucleotide sequences,

wherein said cycle numbers indicate whether MRSA is present in a sample.

2. A method according to claim 1, wherein said first set of primers comprises a plurality of primers with sequences according to at least one of SEQ ID NOs:

10-11.

3. A method according to claim 1, wherein said second set of primers comprises a plurality of primers with sequences according to at least one of SEQ ID NOs:

2-8.

4. A method according to claim 1, wherein said third set of primers comprises a plurality of primers with sequences according to at least one of SEQ ID NOs:

13-14 and 18-19.

5. A method according to claim 1, wherein said amplification reaction is a real-time polymerase chain reaction (PCR).

6. A method according to claim 1, said method further comprising:

(g) contacting said sample with primers complementary to at least a portion of a human gene polynucleotide sequence;

(h) amplifying said human gene polynucleotide sequence; and

(i) detecting said amplified human gene polynucleotide sequence.

7. A method according to claim 1, wherein said S. aureus-specific polynucleotide sequence is at least a portion of a gene, which is a member selected from nuc, Sa442, and femB.

8. A method according to claim 7, wherein said gene is nuc.

9. A method according to claim 7, wherein said gene is Sa442.

10. A method of detecting methicillin-resistant S. aureus (MRSA) in a sample, said method comprising:

(a) determining whether a mecA polynucleotide is present in said sample;

(b) determining whether a bridging region polynucleotide is present in said sample; and

(c) determining whether an S. aureus-specific polynucleotide is present in said sample, wherein said S. aureus-specific polynucleotide is not an orfX polynucleotide,

wherein if said mecA polynucleotide, said bridging region polynucleotide, and said S. aureus-specific polynucleotide are all present in said sample, then MRSA is present in said sample.

11. A method according to claim 10, wherein said determining whether said mecA polynucleotide is present in said sample comprises conducting an amplification reaction on said sample, wherein said amplification reaction utilizes a first set of primers, and wherein a majority of said first set of primers are complementary to at least a portion of said mecA polynucleotide.

12. A method according to claim 11, wherein said first set of primers comprises at least one of SEQ ID NOs. 10-11.

13. A method according to claim 10, wherein said determining whether said orfX polynucleotide is present in said sample comprises conducting an amplification reaction on said sample, wherein said amplification reaction utilizes a second set of primers, and wherein a majority of said second set of primers are complementary to at least a portion of said orfX polynucleotide.

14. A method according to claim 13, wherein said second set of primers comprises at least one of SEQ ID NOs. 2-8.

15. A method according to claim 10, wherein said determining whether said S. aureus-specific polynucleotide is present in said sample comprises conducting an amplification reaction on said sample, wherein said amplification reaction utilizes a third set of primers, and wherein a majority of said third set of primers are complementary to at least a portion of said S. aureus-specific polynucleotide.

16. A method according to claim 15, wherein said third set of primers comprises at least one of SEQ ID NOs. 13-14 and 18-19.

17. A method according to claim 10, wherein said S. aureus-specific polynucleotide is at least a portion of a gene, which is a member selected from nuc, Sa442, and femB.

18. A method according to claim 17, wherein said S. aureus-specific polynucleotide is nuc.

19. A method according to claim 17, wherein said S. aureus-specific polynucleotide is Sa442.

20. A method according to claim 10, said method further comprising:

(d) contacting said sample with primers complementary to at least a portion of a human gene polynucleotide sequence;

(e) amplifying said human gene polynucleotide sequence; and

(f) detecting said amplified human gene polynucleotide sequence.

21. A method according to claim 20, wherein said human gene polynucleotide sequence comprises at least a portion of a housekeeping gene.

22. A method according to claim 20, wherein said human gene polynucleotide sequence comprises at least a portion of a P-globin gene.

23. A method according to claim 10, wherein steps (a), (b), and (c) are performed simultaneously.

24. A method according to claim 10, wherein steps (a), (b), and (c) are performed sequentially in any order.

25. A method according to claim 10, wherein steps (a), (b), and (c) are performed in an identical aliquot of said sample.

26. A method according to claim 10, wherein steps (a), (b), and (c) are performed in different aliquots of said sample.

27. A method according to claim 10, wherein if said mecA polynucleotide is present in said sample and said S. aureus-specific polynucleotide is present in said sample but said bridging region polynucleotide is not present in said sample, then said sample comprises non-Staphylococcus methicillin-resistant bacteria.

28. A method of detecting methicillin-resistant S. aureus (MRSA) in a sample, said method comprising:

(a) providing a first set of primers, wherein said first set of primers are complementary to at least a portion of a mecA polynucleotide sequence;

(b) providing a second set of primers, wherein said second set of primers are complementary to at least a portion of an MSSA-orfX polynucleotide sequence;

(c) providing a third set of primers, wherein said third set of primers are complementary to at least a portion of an S. aureus-specific polynucleotide sequence, and wherein said S. aureus-specific polynucleotide sequence is not an orfX polynucleotide;

(d) combining said first, second and third set of primers with said sample in a reaction mixture;

(e) performing a multi-cycle amplification reaction with said reaction mixture;

(f) determining cycle numbers of appearance of each of said mecA, MSSA-orfX and S. aureus-specific polynucleotide sequences,

wherein said cycle numbers indicate whether MRSA is present in a sample.

29. A method according to claim 28, wherein if said S. aureus-specific polynucleotide sequence appears at a cycle at least 4 cycles removed from appearance of said MSSA-orfX polynucleotide sequence, then said sample comprises both MRSA and methicillin-sensitive S. aureus (MSSA).

30. A method according to claim 28, wherein if said S. aureus-specific polynucleotide sequence appears at a within 3 cycles of said MSSA-orfX polynucleotide sequence, then said sample comprises MSSA and a non-S. aureus methicillin resistant bacteria.

31. A method of identifying bacteria in a sample, said method comprising:

(a) determining whether a mecA polynucleotide is present in said sample;

(b) determining whether an MSSA-orfX polynucleotide is present in said sample; and

(c) determining whether an S. aureus-specific polynucleotide is present in said sample, wherein said S. aureus-specific polynucleotide is not an orfX polynucleotide,

wherein the combination of (a), (b), and (c) present in said sample identifies bacteria in said sample.

32. A method according to claim 31, wherein if said mecA polynucleotide and said S. aureus-specific polynucleotide are both present in said sample and said MSSA-orfX polynucleotide is not present in said sample, then said sample comprises methicillin-resistant S. aureus (MRSA).

33. A method according to claim 31, wherein if said S. aureus-specific polynucleotide and said MSSA-orfX are both present in said sample and said mecA polynucleotide is not present in said sample, then said sample comprises methicillin-susceptible S. aureus (MSSA) but does not comprise MRSA.

34. A method according to claim 31, wherein if said mecA polynucleotide and said MSSA-orfX polynucleotide are both present in said sample and said S. aureus-specific polynucleotide is not present in said sample, then said sample comprises a non-S. aureus methicillin-resistant bacteria and said sample does not comprise MRSA.

35. A method according to claim 31, wherein if only one of said mecA polynucleotide, said MSSA-orfX polynucleotide and said S. aureus-specific polynucleotide are present in said sample, then said sample does not comprise MRSA.

36. A method according to claim 31, wherein said sample may comprise no bacteria, bacteria from a single strain, or a mixture of bacteria from more than one strain.

37. A method according to claim 31, wherein said sample is a member selected from a bodily fluid, a nasal swab, and a tissue.

38. A method according to claim 31, wherein said determining whether a mecA polynucleotide is present in said sample, said determining whether an MSSA-orfX polynucleotide is present in said sample, and said determining whether an S. aureus-specific polynucleotide is present in said sample are all amplification reactions conducted in separate aliquots of said sample.

39. A method according to claim 31, wherein two or more of said determining whether a mecA polynucleotide is present in said sample, said determining whether an MSSA-orfX polynucleotide is present in said sample, and said determining whether an S. aureus-specific polynucleotide is present in said sample are amplification reactions conducted in identical aliquots of said sample.

40. A kit for identifying MRSA in a sample, said kit comprising:

(a) a first set of primers complementary to at least a portion of a mecA polynucleotide sequence;

(b) a second set of primers complementary to at least a portion of MSSA-orfX polynucleotide sequence;

(c) a third set of primers complementary to at least a portion of an S. aureus-specific polynucleotide sequence, wherein said S. aureus-specific polynucleotide sequence is not a bridging sequence; and

(d) at least one member selected from;

a DNA polymerase enzyme, dNTPs, magnesium and a stabilizer.

41. A kit for identifying MRSA in a sample, said kit comprising:

(a) a first set of primers complementary to at least a portion of a mecA polynucleotide sequence;

(b) a second set of primers complementary to at least a portion of a bridging sequence;

(c) a third set of primers complementary to at least a portion of an S. aureus-specific polynucleotide sequence, wherein said S. aureus-specific polynucleotide sequence is not a bridging sequence; and

(d) at least one member selected from;

a DNA polymerase enzyme, dNTPs, magnesium and a stabilizer.

42. A method of detecting an antibiotic-resistant bacterial strain in a sample, said method comprising:

(a) providing a first set of primers, wherein said first set of primers are capable of producing a first amplification product from at least a portion of a gene, wherein said gene confers antibiotic-resistance;

(b) providing a second set of primers, wherein said second set of primers are capable of producing a second amplification product from at least a portion of a bridging region;

(c) providing a third set of primers, wherein said third set of primers are capable of producing a third amplification product from at least a portion of a bacterial strain-specific polynucleotide sequence;

(d) combining said first, second and third set of primers with said sample in a reaction mixture;

(e) performing a multi-cycle amplification reaction with said reaction mixture;

(f) determining cycle numbers of appearance of each of said first, second and third amplification products,

wherein said cycle numbers indicate whether an antibiotic-resistant bacterial strain is present in said sample.

43. A method according to claim 42, wherein said gene confers resistance to vancomycin.

44. A method according to claim 42, wherein said gene confers resistance to methicillin.

45. A method according to claim 42, wherein said bacterial strain-specific polynucleotide sequence is an S. aureus-specific polynucleotide sequence.

46. A method according to claim 42, wherein said bridging region comprises a polynucleotide sequence that is a member selected from SEQ ID NOs:

2-8.