Method for the detection and identification of methicillin-resistant Staphylococcus aureus

• US 9,777,335 B2
• Filed: 05/02/2006
• Issued: 10/03/2017
• Est. Priority Date: 06/04/2001
• Status: Expired due to Term

• Current Assignee: Geneohm Sciences Canada, Inc. (Becton, Dickinson & Co)
• Original Assignee: Geneohm Sciences Canada, Inc. (Becton, Dickinson & Co)
• Inventors: Huletsky, Ann, Rossbach, Valery
• Primary Examiner(s): Johannsen, Diana B
• Application Number:
  US11/416,500
• Publication Number:
  US 20060252078A1
• Time in Patent Office: 4,172 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The present invention describes novel SCCmec right extremity junction sequences for the detection of methicillin-resistant Staphyloccocus aureus (MRSA). It relates to the use of these DNA sequences for diagnostic purposes.

Claims

1. A method to detect the presence of an MREJ type vii methicillin-resistant Staphylococcus aureus (MRSA) strain comprising:

a.) performing an amplification reaction comprising contacting a sample to be analyzed for the presence of said MREJ type vii MRSA strain with a first amplification primer and a second amplification primer to generate a first amplicon if said MREJ type vii MRSA strain is present in said sample, said MREJ type vii MRSA strain including a staphylococcal cassette chromosome mec (SCCmec) element containing a mecA gene inserted into chromosomal DNA, said chromosomal DNA being orfX, thereby generating a polymorphic right extremity junction (MREJ) type vii sequence that comprises sequences from both the SCCmec element right extremity and orfX adjoining said right extremity;

wherein said first primer is at least 10 nucleotides in length and specifically hybridizes at least under conditions of 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂ at 55°

C. with an SCCmec element right extremity of an MREJ nucleic acid type vii sequence selected from the group consisting of;

SEQ ID NO;

165, SEQ ID NO;

166, and the complements thereof,

wherein said second primer is at least 10 nucleotides in length and specifically hybridizes at least under conditions of 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂ at 55°

C. with orfX,

wherein said first amplicon generated if said MREJ type vii MRSA strain is present in said sample comprises polymorphic right extremity junction (MREJ) type vii sequence and orfX sequence, including the junction of the two, and is indicative of the presence of MREJ type vii MRSA strain in said sample; and

b.) detecting said first amplicon if present.

2. The method of claim 1, wherein said first amplification primer that specifically hybridizes with said SCCmec element right extremity of an MREJ type vii sequence comprises at least 10 consecutive residues of SEQ ID NO:

112, or the complement thereof.

3. The method of claim 1, wherein said first amplification primer that specifically hybridizes with said SCCmec element right extremity of an MREJ type vii sequence comprises at least 10 consecutive residues of SEQ ID NO:

113, or the complement thereof.

4. The method of claim 2, wherein said second amplification primer comprises at least ten consecutive residues of SEQ ID NO:

64 or the complement thereof.

5. The method of claim 3, wherein said second amplification primer comprises at least ten consecutive residues of SEQ ID NO:

64 or the complement thereof.

6. The method of claim 1, wherein said amplification reaction comprises PCR.

7. The method of claim 1, wherein said method comprises the use of at least one first or second amplification primer and/or a probe comprising a nucleic acid sequence or complement thereof selected from the group consisting of SEQ ID NOs:

112, 113, 114, 119, 120, 121, 122, 123, 150, 151, 153, 64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159, 59, 62, 32, 83, 84, 160, 161, 162, 163, and 164 for the detection of MREJ type vii.

8. The method of claim 1, wherein said second and first amplification primers are a primer pair consisting of SEQ ID NOs:

64 and 112 or SEQ ID NOs;

64 and 113, or the complements thereof.

9. The method of claim 8, further comprising the use of at least one probe having a sequence selected from the group consisting of SEQ ID NOs:

32, 83, 84, 160, 161, 162, 163, 164, and the complements thereof.

10. The method of claim 1, wherein said method comprises the use of at least one first or second amplification primer and/or a probe comprising a nucleic acid sequence or complement thereof selected from the group consisting of:

SEQ ID NOs;

64, 112, 113, 84, 163, and 164, for the detection of MREJ type vii.

11. The method of claim 1, further comprising detecting the presence of at least one further methicillin-resistant Staphylococcus aureus (MRSA) strain in said sample, said at least one further MRSA strain including an SCCmec element containing a mecA gene inserted into chromosomal DNA, thereby generating a polymorphic right extremity junction (MREJ) type i, ii, iii, iv, v, vi, viii or ix nucleic acid sequence that comprises sequences from both the SCCmec element right extremity and chromosomal DNA adjoining said right extremity, wherein said method further comprises contacting said sample with at least one additional primer to generate a second amplicon if said at least one further MREJ type i, ii, iii, iv, v, vi, viii or ix MRSA strain is present in said sample,

wherein said at least one additional primer is at least 10 nucleotides in length and specifically hybridizes at least under conditions of 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂ at 55°

C. with said polymorphic sequences from the SCCmec element right extremity of said at least one of MREJ type i, ii, iii, iv, v, vi, viii and ix nucleic acid sequences, or complements thereof, selected from the group consisting of;

a) SEQ ID NOs;

1, 20-25, and 41 for MREJ type i;

b) SEQ ID NOs;

2, 17-19, 26, 40, 173-183, 185, 186 and 197 for MREJ type ii;

c) SEQ ID NOs;

4-16, 104, 184 and 198 for MREJ type iii;

d) SEQ ID NOs;

42-46 and 51 for MREJ type iv;

e) SEQ ID NOs;

47-50 for MREJ type v;

f) SEQ ID NO;

171 for MREJ type vi;

g) SEQ ID NO;

167 for MREJ type viii; and

h) SEQ ID NO;

168 for MREJ type ix,

wherein said second amplicon generated if said at least one further MREJ type i, ii, iii, iv, v, vi, viii or ix MRSA strain is present in said sample comprises polymorphic right extremity junction (MREJ) type i, ii, iii, iv, v, vi, viii or ix sequence and orfX sequence, including the junction of the two, and is indicative of the presence of said at least one further MREJ type i, ii, iii, iv, v, vi, viii or ix MRSA strain in said sample; and

detecting said second amplicon if present.

12. The method of claim 11, wherein said at least one additional primer comprises at least one primer selected from the following SEQ ID NOs or complements thereof:

66, 100, 101, 105, 52, 53, 54, 55, 56, 57, 97, 99, 106, 117, 118, 124, 125, 58, 67, 98, 102, 107, 108, 79, 77, 145, 146, 147, 65, 80, 154, 155, 202, 203, 204, 115, 116, 187, 188, 207, 208, 109, 148, 149, 205, and 206.

13. The method of claim 12, further comprising use of at least one second amplification primer and/or a probe selected from the following SEQ ID NOs or complements thereof:

64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159, 59, 62, 32, 83, 84, 160, 161, 162, 163, and 164.

14. The method of claim 11, further comprising the use of at least one primer pair selected from the following SEQ ID NOs, or the complements thereof:

a) 64/66, 64/100, 64/101, 59/52, 59/53, 59/54, 59/55, 59/56, 59/57, 60/52, 60/53, 60/54, 60/55, 60/56, 60/57, 61/52, 61/53, 61/54, 61/55, 61/56, 61/57, 62/52, 62/53, 62/54, 62/55, 62/56, 62/57, 63/52, 63/53, 63/54, 63/55, 63/56 and 63/57, for the detection of type i MREJ;

b) 64/66, 64/97, 64/99, 64/100, 64/101, 59/52, 59/53, 59/54, 59/55, 59/56, 59/57, 60/52, 60/53, 60/54, 60/55, 60/56, 60/57, 61/52, 61/53, 61/54, 61/55, 61/56, 61/57, 62/52, 62/53, 62/54, 62/55, 62/56, 62/57, 63/52, 63/53, 63/54, 63/55, 63/56 and 63/57, for the detection of type ii MREJ;

c) 64/67, 64/98, 64/102, 59/58, 60/58, 61/58, 62/58 and 63/58, for the detection of type iii MREJ;

d) 64/79, for the detection of type iv MREJ;

e) 64/80, for the detection of type v MREJ;

f) 64/204, for the detection of type vi MREJ;

g) 64/115 and 64/116, for the detection of type viii MREJ; and

h) 64/109, for the detection of type ix MREJ.

15. The method of claim 14, comprising the use of at least one probe having a sequence, or the complement thereof, selected from the group consisting of:

SEQ ID NOs;

32, 83, 84, 160, 161, 162, 163 and 164.

16. The method of claim 11, comprising the use of primers and probes having the following nucleotide sequences, or the complements thereof:

a) SEQ ID NOs;

64, 66, and at least one of 84, 163, 164 for the detection of MREJ type i or ii;

b) SEQ ID NOs;

64, 67, and at least one of 84, 163, 164 for the detection of MREJ type iii;

c) SEQ ID NOs;

64, 79, and at least one of 84, 163, 164 for the detection of MREJ type iv; and

d) SEQ ID NOs;

64, 80, and at least one of 84, 163, 164 for the detection of MREJ type v.

17. The method of claim 11, wherein multiple primers and/or probes are used together in the same physical enclosure.

18. The method of claim 11, further comprising distinctively detecting each amplicon if present as an indication of the presence of said MREJ type vii and said at least one further MREJ type selected from MREJ types i, ii, iii, iv, v, vi, viii and ix, wherein the presence or absence of an amplicon produced by a primer is indicative of the presence or absence, respectively, of the corresponding MREJ type MRSA.

19. The method of claim 11, wherein a plurality of primers and/or probes all chosen to hybridize under the same hybridization conditions are used.

20. The method of claim 1, comprising detecting the presence or absence of at least three further MRSA strains in said sample, said at least three further MRSA strain including an SCCmec element containing a mecA gene inserted into chromosomal DNA, thereby generating a polymorphic right extremity junction (MREJ) type i, ii, iii, iv, v, vi, viii or ix nucleic acid sequence that comprises sequences from both the SCCmec element right extremity and chromosomal DNA adjoining said right extremity, wherein said method further comprises contacting said sample with at least three additional primers to generate a second, third or fourth amplicon if said at least three further MREJ type i, ii, iii, iv, v, vi, viii or ix MRSA strains are present in said sample,

wherein said at least three additional primers are at least 10 nucleotides in length and each specifically hybridizes under conditions of 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂ at 55°

C. with said polymorphic sequences from the SCCmec element right extremity of one of said at least three further MRSA strains of MREJ type i-vi, and viii-ix nucleic acid sequences, or complements thereof, selected from the group consisting of;

a) SEQ ID NOs;

1, 20-25, and 41 for MREJ type i;

b) SEQ ID NOs;

2, 17-19, 26, 40, 173-183, 185, 186 and 197 for MREJ type ii;

c) SEQ ID NOs;

4-16, 104, 184 and 198 for MREJ type iii;

d) SEQ ID NOs;

42-46 and 51 for MREJ type iv;

e) SEQ ID NOs;

47-50 for MREJ type v;

f) SEQ ID NO;

171 for MREJ type vi;

g) SEQ ID NO;

167 for MREJ type viii; and

h) SEQ ID NO;

168 for MREJ type ix

wherein said second, third and fourth amplicon generated if said at least three further MREJ type i, ii, iii, iv, v, vi, viii or ix MRSA strains are present in said sample comprises polymorphic right extremity junction (MREJ) type i, ii, iii, iv, v, vi, viii or ix sequence and orfX sequence, including the junction of the two, and said second, third and fourth amplicons are indicative of the presence of said at least three further MREJ type i, ii, iii, iv, v, vi, viii or ix MRSA strains in said sample; and

detecting the presence or absence of each amplicon distinctively,

wherein the presence or absence of an amplicon produced by a primer is indicative of the presence or absence, respectively, of the corresponding MREJ type MRSA, thereby determining the presence or absence in a sample of at least four MREJ types of MRSA, one of which is MREJ type vii.

21. The method of claim 20, for determining the presence or absence of MREJ type vii, type i, type ii, and type iii.

22. The method of claim 20, for determining the presence or absence of MREJ type vii, type i, type ii, type iii and type iv.

23. The method of claim 20, for determining the presence or absence of MREJ type vii, type i, type ii, type iii, type iv, type v, type vi, type viii and type ix.

24. The method of claim 20, for determining the presence or absence of MREJ type vii, type i, type ii, type iii, type iv and type vi.

25. The method of claim 1, wherein said method comprises the use of at least one second amplification primer and/or a probe specific for the S. aureus chromosome comprising a sequence, or the complement thereof, selected from the group consisting of SEQ ID NOs:

32, 59, 62, 70-76, 83, 84, 103, 130, 132, and 160-164.

26. The method of claim 11, wherein multiplex PCR is used.

27. The method of claim 1, comprising the use of at least one probe having a sequence, or the complement thereof, selected from the group consisting of SEQ ID NOs:

84, 163 and 164.

28. The method of claim 1, wherein said method comprises the use of at least one first amplification primer comprising a nucleic acid sequence or complement thereof selected from the group consisting of SEQ ID NOs:

112, 113, 114, 119, 120, 121, 122, 123, 150, 151, and 153, for the detection of MREJ type vii.

29. The method of claim 28, wherein said method further comprises the use of at least one second amplification primer and/or probe comprising a nucleic acid sequence or complement thereof selected from the group consisting of SEQ ID NOs:

64, 71, 72, 73, 74, 75, 76, 70, 103, 130, 132, 158, 159, 59, 62, 32, 83, 84, 160, 161, 162, 163, and 164.