Methods, compositions and kits for detection and analysis of antibiotic-resistant bacteria

• US 8,557,524 B2
• Filed: 08/08/2011
• Issued: 10/15/2013
• Est. Priority Date: 04/19/2007
• Status: Expired due to Fees

• Current Assignee: Molecular Detection Inc.
• Original Assignee: Molecular Detection Inc.
• Inventors: Paitan, Yosef
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US13/205,496
• Publication Number:
  US 20110294695A1
• Time in Patent Office: 799 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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 methiceillin-resistant S. aureus (MRSA) in a sample, wherein said sample contains one or a mixture of bacterial species, said method comprising:

(a) amplifying a mecA polynucleotide sequence to produce an amplified mecA polynucleotide sequence;

(b) determining a cycle threshold (CT) value of said amplified mecA polynucleotide sequence;

(c) amplifying a bridging region polynucleotide sequence from said sample to produce an amplified bridging region polynucleotide sequence;

(d) determining a CT value of said amplified bridging region polynucleotide sequence;

(e) amplifying an S. aureus-specific polynucleotide sequence to produce an amplified S. aureus-specific polynucleotide sequence;

(f) determining a CT value of said amplified S. aureus-specific polynucleotide sequence, wherein said S. aureus-specific polynucleotide sequence is not an orfX polynucieotide sequence,

(g) comparing said CT values of said amplified mecA, amplified bridging region, and amplified S. aureus-specific polynucleotide sequences to each other,

wherein if said CT values of each of said amplified mecA polynueleotide, said amplified bridging region polynueleotide, and said amplified S. aureus-specific polynucleotide sequences are within a predetermined range to each other, then MRSA is present in said sample.

2. A method according to claim 1, wherein said mecA polynucleotide is amplified from said sample using a first set of primers.

3. A method according to claim 2, wherein said first set of primers comprises at least one of SEQ ID NOs. 10-11.

4. A method according to claim 1, wherein said bridging region polynucleotide is amplified from said sample using a second set of primers.

5. A method according to claim 4, wherein said second set of primers comprises at least one of SEQ ID NOs. 2-8.

6. A method according to claim 1, wherein said S. aureus-specific polynucleotide is amplified from said sample using a third set of primers.

7. A method according to claim 6, wherein said third set of primers comprises at least one of SEQ ID NOs. 13-14 and 18-19.

8. A method according to claim 1, wherein said S. aureus-specific polynucleotide is a member selected from nuc and Sa442.

9. A method according to claim 8, wherein said S. aureus-specific polynucleotide is nuc.

10. A method according to claim 8, wherein said S. aureus-specific polynucleotide is Sa442.

11. A method according to claim 1, wherein steps (a), (c), and (e) or steps (b), (d), and (f) are performed simultaneously.

12. A method according to claim 1, wherein steps (a), (c), and (e) or steps (b), (d), and (f) are performed sequentially in any order.

13. A method according to claim 1, wherein steps (a) through (e) are performed in an aliquot of said sample.

14. A method according to claim 1, wherein steps (a) and (b), steps (c) and (d), and steps (e) and (f) are performed in different aliquots of said sample.

15. A method according to claim 1, wherein said predetermined range is 3 CT values.