Methods, compositions and kits for detection and analysis of antibiotic-resistant bacteria
First Claim
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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 an MSSA-orfX polynucleotide sequence, wherein said MSSA-orfX polynucleotide sequence is from a region of the orfX gene surrounding the position at which an insertion cassette would be inserted in methicillin resistant S. aureus but is not inserted in methicillin susceptible S. aureus;
(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.
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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.
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Citations
13 Claims
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1. A method of detecting methicillin-resistant S. aureus (MRSA) in a sample, said method comprising:
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(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, wherein said MSSA-orfX polynucleotide sequence is from a region of the orfX gene surrounding the position at which an insertion cassette would be inserted in methicillin resistant S. aureus but is not inserted in methicillin susceptible S. aureus;
(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. - View Dependent Claims (2, 3)
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4. A method of identifying bacteria in a sample, said method comprising:
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(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, wherein said MSSA-orfX polynucleotide sequence is from a region of the orfX gene surrounding the position at which an insertion cassette would be inserted in methicillin resistant S. aureus but is not inserted in methicillin susceptible S. aureus;
(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 whether a mecA polynucleotide is present in said sample; (g) determining whether an MSSA orfX polynucleotide is present in said sample; and (h) 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 (f), (g), and (h) present in said sample identifies bacteria in said sample. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12)
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13. A kit for identifying MRSA in a sample, said kit comprising:
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(a) a first set of primers complementary to at least a portion of a mecA polynucleotide sequence; (b) a second set of printers complementary to at least a portion of MSSA-orfX polynucleotide sequence, wherein said MSSA-orfX polynucleotide sequence is from a region of the orfX gene surrounding the position at which an insertion cassette would be inserted in methicillin resistant S. aureus but is not inserted in methicillin susceptible S. aureus;
(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.
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Specification