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

• US 10,577,664 B2
• Filed: 09/18/2017
• Issued: 03/03/2020
• 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:
  US15/707,421
• Publication Number:
  US 20180208972A1
• Time in Patent Office: 897 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 v methicillin-resistant Staphylococcus aureus (MRSA) strain nucleic acid in a sample, comprising:

a) performing an amplification reaction comprising contacting a sample to be analyzed for the presence of said MREJ type v MRSA strain nucleic acid with a first amplification primer and a second amplification primer to generate a first amplicon if said MREJ type v MRSA strain nucleic acid is present in said sample, said MREJ type v MRSA strain nucleic acid 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 v nucleic acid sequence that comprises nucleic acid 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 type v nucleic acid sequence selected from the group consisting of;

SEQ ID NOs;

47-50, 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 v MRSA strain nucleic acid is present in said sample comprises polymorphic right extremity junction (MREJ) type v sequence and orfX sequence, including the junction of the two, and is indicative of the presence of MREJ type v MRSA strain nucleic acid in said sample; and

b) generating and detecting said first amplicon.

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

80, or the complement thereof.

3. 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.

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

5. 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:

65, 80, 154, 155, 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 v nucleic acid.

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

64 and 80, or the complements thereof.

7. The method of claim 6, further comprising the use of at least one probe having a nucleic acid sequence selected from the group consisting of SEQ ID NOs:

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

8. 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, 80, 84, 163, and 164, for the detection of MREJ type v nucleic acid.

9. The method of claim 1, further comprising detecting the presence of at least one further methicillin-resistant Staphylococcus aureus (MRSA) strain nucleic acid 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, vi, viii or ix nucleic acid sequence that comprises nucleic acid 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, vi, viii or ix MRSA strain nucleic acid 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 nucleic acid sequences from the SCCmec element right extremity of said at least one of MREJ type i, ii, iii, iv, 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 NO;

171 for MREJ type vi;

f) SEQ ID NO;

167 for MREJ type viii; and

g) 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, vi, viii or ix MRSA strain nucleic acid is present in said sample comprises polymorphic right extremity junction (MREJ) type i, ii, iii, iv, 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, vi, viii or ix MRSA strain nucleic acid in said sample; and

detecting said second amplicon if present.

10. The method of claim 9, 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, 202, 203, 204, 114, 119, 120, 121, 122, 123, 150, 151, 153, 115, 116, 187, 188, 207, 208, 109, 148, 149, 205, and 206.

11. The method of claim 10, 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.

12. The method of claim 9, 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 nucleic acid;

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 nucleic acid;

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 nucleic acid;

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

e) 64/204, for the detection of type vi MREJ nucleic acid;

f) 64/115 and 64/116, for the detection of type viii MREJ nucleic acid; and

g) 64/109, for the detection of type ix MREJ nucleic acid.

13. The method of claim 12, comprising the use of at least one probe having a nucleic acid sequence, or the complement thereof, selected from the group consisting of:

SEQ ID NOs;

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

14. The method of claim 9, comprising the use of primers and probes having the following nucleic acid 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 nucleic acid;

b) SEQ ID NOs;

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

c) SEQ ID NOs;

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

15. The method of claim 9, wherein multiple primers and/or probes are used together in the same physical enclosure.

16. The method of claim 9, further comprising distinctively detecting said first amplicon as an indication of the presence of said MREJ type v nucleic acid and said second amplicon if present as an indication of said at least one further MREJ type nucleic acid selected from MREJ types i, ii, iii, iv, vi, viii and ix, wherein the presence or absence of said second amplicon produced by a primer is indicative of the presence or absence, respectively, of the corresponding MREJ type i, ii, iii, iv, vi, viii or ix MRSA nucleic acid.

17. The method of claim 9, wherein a plurality of primers and/or probes all chosen to hybridize under the same hybridization conditions are used.

18. The method of claim 1, comprising detecting the presence or absence of at least three further MRSA strain nucleic acids in said sample, said at least three further MRSA strain nucleic acids 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, vi, viii or ix nucleic acid sequence that comprises nucleic acid 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, vi, viii or ix MRSA strain nucleic acids 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 nucleic acid sequences from the SCCmec element right extremity of one of said at least three further MRSA strains of MREJ type i-iv, 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 NO;

171 for MREJ type vi;

f) SEQ ID NO;

167 for MREJ type viii; and

g) 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, vi, viii or ix MRSA strain nucleic acids are present in said sample comprises polymorphic right extremity junction (MREJ) type i, ii, iii, iv, vi, viii or ix nucleic acid 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, vi, viii or ix MRSA strain nucleic acids in said sample; and

detecting the presence or absence of each second, third and/or fourth amplicon distinctively,

wherein the presence or absence of each second, third and/or fourth amplicon produced by a primer is indicative of the presence or absence, respectively, of the corresponding one of said three further MREJ type MRSA nucleic acids, thereby determining the presence or absence in a sample of at least three further MREJ types of MRSA nucleic acids.

19. The method of claim 18, for further determining the presence or absence of MREJ type i, type ii, and type iii nucleic acids.

20. The method of claim 18, for further determining the presence or absence of MREJ type i, type ii, type iii and type iv nucleic acids.

21. The method of claim 18, for further determining the presence or absence of MREJ type i, type ii, type iii, type iv, type vi, type type viii and type ix nucleic acids.

22. The method of claim 18, for further determining the presence or absence of MREJ type i, type ii, type iii, type iv and type vi nucleic acids.

23. 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 nucleic acid 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.

24. The method of claim 9, wherein multiplex PCR is used.

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

84, 163 and 164.

26. 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:

65, 80, 154, 155, for the detection of MREJ type v nucleic acid.

27. The method of claim 26, 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.