Sequences for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA)

• US 20070082340A1
• Filed: 10/11/2005
• Published: 04/12/2007
• Est. Priority Date: 10/11/2005
• Status: Active Grant

• Current Assignee: Geneohm Sciences Incorporated (Becton, Dickinson & Co)
• Original Assignee: Geneohm Sciences Incorporated (Becton, Dickinson & Co)
• Inventors: Giroux, Richard, Huletsky, Ann
• Granted Patent: US 7,838,221 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

Described herein are novel SCCmec right extremity junction (MREJ) sequences for the detection and/or identification of methicillin-resistant Staphylococcus aureus (MRSA). Disclosed are methods and compositions based on DNA sequences for the specific detection of MREJ sequences designated types xi, xii, xiii, xiv, xv, xvi, xvii, xviii, xix, and xx for diagnostic purposes and/or epidemiological typing.

Claims

1. A method of detecting the presence of a methicillin-resistant Staphylococcus aureus (MRSA) strain in a sample comprising nucleic acids, wherein the MRSA strain comprises an SCCmec insert comprising a mecA gene inserted in bacterial nucleic acids, and wherein the insertion of said SSCmec insert in the bacterial nucleic acids generates a polymorphic right extremity junction (MREJ), said method comprising:

providing at least one primer and/or probe that is specific for MRSA strains, wherein the primer and/or probe anneals to at least one polymorphic MREJ nucleic acid of types xi to xx;

annealing the nucleic acids of the sample with the primer(s) and/or probe(s), wherein annealed primer(s) and/or probe(s) indicate the presence of an MRSA strain in the sample; and

detecting the presence and/or amount of annealed probe(s), or detecting the amount of an amplification product produced through annealing of the primers to the nucleic acids, as an indication of the presence and/or amount of MRSA.

2. The method of claim 1, wherein the providing step further comprises providing a plurality of primers and/or probes that can anneal with at least one MREJ type selected from MREJ type xi, xii, xiii, xiv, xv, xvi, xvii, xviii, xix, and xx.

3. The method of claim 2, further comprising a DNA amplification step, wherein the DNA amplification is PCR, LCR, NABSA, 3SR, SDA, bDNA, TMA, CPT, SPA, NDSA, rolling circle amplification, anchored strand displacement amplification, solid phase (immobilized) rolling circle amplification, or Q beta replicase amplification.

4. The method of claim 2, wherein the plurality of primers and/or probes can anneal with at least four MREJ types selected from MREJ type xi, xii, xiii, xiv, xv, xvi, xvii, xviii, xix, and xx.

5. The method of claim 2, wherein the primers and/or probes anneal under common annealing conditions.

6. The method of claim 5, wherein the primer and/or probes are provided in the same physical enclosure.

7. The method of claim 1, wherein the primers and/or probes are at least 10, 12, 14, 16, 18, 20, 25, or 30 nucleotides in length.

8. The method of claim 1, wherein the primers and/or probes are capable of annealing to at least one of SEQ ID NOs:

15, 16, 17, 18, 19, 20, 21, 25, 26, 39, 40, 41, 42, 55, and 56.

9. The method of claim 2, wherein at least one primer and/or probe of the plurality of primers and/or probes is capable of annealing to each of SEQ ID NOs:

15, 16, 17, 18, 19, 20, 21, 25, 26, 39, 40, 41, 42, 55, and 56.

10. The method of claim 2, wherein the plurality of primers and/or probes comprises SEQ ID NOs:

51, 30, 31, 32, and 33 for the detection of MREJ type xi.

11. The method of claim 2, wherein the plurality of primers and/or probes comprises SEQ ID NOs:

52, 30, 31, 32, and 33, for the detection of MREJ type xii.

12. The method of claim 2, wherein the plurality of primers and/or probes comprises SEQ ID NOs:

29, 30, 31, 32, and 33 for the detection of MREJ type xiii.

13. The method of claim 2, wherein the plurality of primers and/or probes comprises SEQ ID NOs:

29, 30, 31, 32, and 33 for the detection of MREJ type xiv.

14. The method of claim 2, wherein the plurality of primers and/or probes comprises SEQ ID NOs:

24, 30, 31, 32, and 33 for the detection of MREJ type xv.

15. The method of claim 2, wherein the plurality of primers and/or probes comprises SEQ ID NOs:

36 and 44 for the detection of MREJ type xvi.

16. The method of claim 2, wherein the plurality of primers and/or probes comprises SEQ ID NOs:

4, 30, 31, 32, and 33 for the detection of MREJ type xvii.

17. The method of claim 2, wherein the plurality of primers and/or probes comprises SEQ ID NOs:

7, 30, 31, 32, and 33 for the detection of MREJ type xviii.

18. The method of claim 2, wherein the plurality of primers and/or probes comprises SEQ ID NOs:

9, 30, 31, 32, and 33 for the detection of MREJ type xix.

19. The method of claim 2, wherein the plurality of primers and/or probes comprises SEQ ID NOs:

8, 30, 31, 32, and 33 for the detection of MREJ type xx.

20. The method of claim 2, wherein the plurality of primers and/or probes comprises

SEQ ID NOs:

51, 30, 31, 32, and 33 for the detection of MREJ type xi,

SEQ ID NOs;

52, 30, 31, 32, and 33 for the detection of MREJ type xii,

SEQ ID NOs;

29, 30, 31, 32, and 33 for the detection of MREJ type xiii

SEQ ID NOs;

29, 30, 31, 32, and 33 for the detection of MREJ type xiv,

SEQ ID NOs;

24, 30, 31, 32, and 33 for the detection of MREJ type xv

SEQ ID NOs;

36 and 44 for the detection of MREJ type xvi,

SEQ ID NOs;

4, 30, 31, 32, and 33 for the detection of MREJ type xvii,

SEQ ID NOs;

7, 30, 31, 32, and 33 for the detection of MREJ type xviii,

SEQ ID NOs;

9, 30, 31, 32, and 33 for the detection of MREJ type xix, and

SEQ ID NOs;

8, 30, 31, 32, and 33 for the detection of MREJ type xx.

21. The method of claim 10, wherein the plurality of primers and/or probes further comprises the primers and/or probes of SEQ ID NOs:

34, 44, 45, and 76 for the detection of MREJ type xi.

23. The method of claim 12, wherein the plurality of primers and/or probes further comprises the primers and/or probes of SEQ ID NOs:

33, 44, 45, and 76, for the detection of MREJ type xiii.

24. The method of claim 13, wherein the plurality of primers and/or probes further comprises the primers and/or probes of SEQ ID NOs:

44, 45, and 69 for the detection of MREJ type xiv.

25. The method of claim 14, wherein the plurality of primers and/or probes further comprises the primers and/or probes of SEQ ID NOs:

4, 45, and 62 for the detection of MREJ type xv.

26. The method of claim 16, wherein the plurality of primers and/or probes further comprises the primers and/or probes of SEQ ID NOs:

44, 45, and 62 for the detection of MREJ type xvii.

27. The method of claim 17, wherein the plurality of primers and/or probes further comprises the primers and/or probes of SEQ ID NOs:

44, 45, and 59 for the detection of MREJ type xviii.

28. The method of claim 18, wherein the plurality of primers and/or probes further comprises the primers and/or probes of SEQ ID NOs:

44, 45, and 59 for the detection of MREJ type xix.

29. The method of claim 19, wherein the plurality of primers and/or probes further comprises the primers and/or probes of SEQ ID NOs:

44, 45, and 59 for the detection of MREJ type xx.

30. The method of claim 20, further comprising the primers and/or probes of SEQ ID NOs:

34, 35, 44, 45, 52, 59, 62, 76.

31. The method of claim 21, wherein the primers are provided in pairs, wherein the primer pair is selected from the group consisting of:

SEQ ID NOs ;

34/45, 34/30, 34/76, and 34/44, for the detection of MREJ type xi.

33. The method of claim 23, wherein the primers are provided in pairs, wherein the primer pair is selected from the group consisting of:

SEQ ID NOs;

29/45, 29/30, 29/76, and 29/44, for the detection of MREJ type xiii.

34. The method of claim 24, wherein the primers are provided in pairs, wherein the primer pair is selected from the group consisting of:

SEQ ID NOs;

29/45, 29/30, 29/59, and 29/44, for the detection of MREJ type xiv.

35. The method of claim 25, wherein the primers are provided in pairs, wherein the primer pair is selected from the group consisting of:

SEQ ID NOs;

24/45, 24/30, 24/62, and 24/44, for the detection of MREJ type xv.

36. The method of claim 26, wherein the primers are provided in pairs, wherein the primer pair is selected from the group consisting of:

SEQ ID NOs;

4/45, 430, 4/62, and 4/44 for the detection of MREJ type xvii.

37. The method of claim 27, wherein the primers are provided in pairs, wherein the primer pair is selected from the group consisting of:

SEQ ID NOs;

7/45, 7/30, 7/59, and 7/44 for the detection of MREJ type xviii.

38. The method of claim 28, wherein the primers are provided in pairs, wherein the primer pair is selected from the group consisting of:

SEQ ID NOs;

9/45, 9/30, 9/59, and 9/44 for the detection of MREJ type xix.

39. The method of claim 29, wherein the primers are provided in pairs, wherein the primer pair is selected from the group consisting of:

SEQ ID NOs;

8/45, 8/30, 8/59, and 8/44 for the detection of MREJ type xx.

40. The method of claim 30, wherein the primers are provided in pairs, wherein the primer pair is selected from the group consisting of:

SEQ ID NOs;

34/45, 34/30, 34/76, and 34/44, for the detection of MREJ type xi;

SEQ ID NOs;

35/45, 35/30, 35/62, and 35/44for the detection of MREJ type xii;

SEQ ID NOs;

29/45, 29/30, 29/76, and 29/44 for the detection of MREJ type xiii;

SEQ ID NOs;

29/45, 29/30, 29/59, and 29/44 for the detection of MREJ type xiv;

SEQ ID NOs;

24/45, 24/30, 24/62, and 24/44 for the detection of MREJ type xv;

SEQ ID NOs;

36/44 for the detection of MREJ type xvi;

SEQ ID NOs;

4/45, 4/30, 4/62, and 4/44 for the detection of MREJ type xvii;

SEQ ID NOs;

7/45, 7/30, 7/59, and 7/44 for the detection of MREJ type xviii;

SEQ ID NOs;

9/45, 9/30, 9/59, and 9/44 for the detection of MREJ type xix; and

SEQ ID NOs;

8/45, 8/30, 8/59, and 8/44 for the detection of MREJ type xx.

41. The method of claim 40, further comprising probes having the following sequences:

SEQ ID NOs;

30, 31, and 32 for the detection of MREJ types to xi to xx.

42. The method of claim 40, wherein said probes and primers are used together.

43. The method of claim 42, wherein said probes and/or primers are used together in the same physical enclosure.

44. The method of claim 1, further comprising detecting an annealed probe and/or primer as an indication of the presence of a determined MREJ type from type xi to xx.

45. The method of claim 44, wherein the method of detection is selected from the group comprising:

agarose gel electrophoresis, fluorescence resonance energy transfer, TaqMan probe, molecular beacon probe, Scorpion probe, nanoparticle probe, Ampliflour probe, chemiluminscense, potentiometry, mass spectrometry, plasmon resonance, polarimetry, colorimetry, flow cytometry, scanometry, and DNA sequencing.

50. A kit comprising a plurality of primers and/or probes, wherein the primers and/or probes are at least 10, 12, 14, 16, 18, 20, 25, or 30 nucleotides in length, and wherein the primers and/or probes hybridize under stringent conditions to at least one of the nucleic acids of claim 13, and wherein the plurality of primers and/or probes hybridizes with at least one MREJ of types selected from xi to xx.

51. The kit of claim 50, further comprising primers and/or probes, which hybridize with one or more MREJ of types selected from i to x.

52. The kit of claim 51, further comprising primer pairs, wherein the primer pairs comprise the nucleotide sequences of SEQ ID NOs:

30/36 for the detection of MREJ type i;

30/36 for the detection of MREJ type ii;

30/70 for the detection of MREJ type iii;

30/71 for the detection of MREJ type iv;

30/72 for the detection of MREJ type v;

30/65 for the detection of MREJ type vi;

30/74 for the detection of MREJ type vii;

30/75 for the detection of MREJ type viii;

30/29 for the detection of MREJ type ix;

73/77 for the detection of MREJ type x;

34/45, 34/30, 34/76, 34/44 for the detection of MREJ type xi;

35/45, 35/30, 35/62, 35/44 for the detection of MREJ type xii;

29/45, 29/30, 29/76, 29/44 for the detection of MREJ type xiii;

29/45, 29/30, 29/59, 29/44 for the detection of MREJ type xiv;

24/45, 24/30, 24/62, 24/44 for the detection of MREJ type xv;

36/44 for the detection of MREJ type xvi;

4/45, 4/30, 4/62, 4/44 for the detection of MREJ type xvii;

7/45, 7/30, 7/59, 7/44 for the detection of MREJ type xviii;

9/45, 9/30, 9/59, 9/44 for the detection of MREJ type xix; and

8/45, 8/30, 8/59, 8/44 for the detection of MREJ type xx.

53. The kit of claim 51, further comprising probes, wherein the probes comprise the nucleic acid sequence of SEQ ID NOs:

31, 32, 33.

22. The method of claim I 1, wherein the plurality of primers and/or probes further comprises the primers and/or probes of SEQ ID NOs:

34, 44, 45, 52, and 62 for the detection of MREJ type xii.

32. The method of claim 22, wherein the primers are provided in pairs, wherein the primer pair is selected from the group consisting of:

SEQ ID NOs ;

35/45, 35/30, 35/62, and 35/44, for the detection of MREJ type xii.

46. A nucleic acid selected from the group consisting of:

SEQ ID NOs;

17, 18, 19 (MREJ type xi);

SEQ ID NO;

20 (MREJ type xii);

SEQ ID NOs;

15, 25, 26 (MREJ type xiii);

SEQ ID NO;

16 (MREJ type xiv);

SEQ ID NO;

56 (MREJ type xv);

SEQ ID NO;

21 (MREJ type xvi);

SEQ ID NO;

55 (MREJ type xvii);

SEQ ID NOs;

39, 40 (MREJ type xviii);

SEQ ID NO;

41 (MREJ type xix); and

SEQ ID NO;

42 (MREJ type xx), or the complement thereof.

47. An oligonucleotide of at least 10 nucleotides in length which hybridizes under stringent conditions with a nucleic acid of claim 46.

48. An oligonucleotide pair selected from the group consisting of:

oligonucleotides comprising the sequences of SEQ ID NOs;

34/45, 34/30, 34/76, and 34/44, for the detection of MREJ type xi,

oligonucleotides comprising the sequences of SEQ ID NOs;

35/45, 35/30, 35/62, and 35/44 for the detection of MREJ type xii,

oligonucleotides comprising the sequences of SEQ ID NOs;

29/45, 29/30, 29/76, and 29/44 for the detection of MREJ type xiii,

oligonucleotides comprising the sequences of SEQ ID NOs;

29/45, 29/30, 29/59, and 29/44 for the detection of MREJ type xiv,

oligonucleotides comprising the sequences of SEQ ID NOs;

24/45, 24/30, 24/62, and 24/44 for the detection of MREJ type xv,

oligonucleotides comprising the sequences of SEQ ID NOs;

36/44 for the detection of MREJ type xvi,

oligonucleotides comprising the sequences of SEQ ID NOs;

4/45, 430, 4/62, and 4/44 for the detection of MREJ type xvii,

oligonucleotides comprising the sequences of SEQ ID NOs;

7/45, 7/30, 7/59, and 7/44 for the detection of MREJ type xviii,

oligonucleotides comprising the sequences of SEQ ID NOs;

9/45, 9/30, 9/59, and 9/44 for the detection of MREJ type xix, and

oligonucleotides comprising the sequences of SEQ ID NOs;

8/45, 8/30, 8/59, and 8/44 for the detection of MREJ type xx.

49. An oligonucleotide comprising the sequence of SEQ ID NO:

31, 32, or 33.