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, wherein said sample contains one or a mixture of bacterial species, 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 a bridging region;
(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, bridging region and S. aureus-specific polynucleotide sequences,(g) comparing said cycle numbers of appearance of said mecA, bridging region, and S. aureus-specific polynucleotide sequences to each other,wherein if said mecA polynucleotide sequence has a cycle number of appearance that is substantially the same as that of either said bridging region sequence or said S. aureus-specific polynucleotide sequence, then MRSA is present in said 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.
131 Citations
33 Claims
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1. A method of detecting methicillin-resistant S. aureus (MRSA) in a sample, wherein said sample contains one or a mixture of bacterial species, 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 a bridging region; (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, bridging region and S. aureus-specific polynucleotide sequences, (g) comparing said cycle numbers of appearance of said mecA, bridging region, and S. aureus-specific polynucleotide sequences to each other, wherein if said mecA polynucleotide sequence has a cycle number of appearance that is substantially the same as that of either said bridging region sequence or said S. aureus-specific polynucleotide sequence, then MRSA is present in said sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method of detecting methicillin-resistant S. aureus (MRSA) in a sample, wherein said sample contains one or a mixture of bacterial species, said method comprising:
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(a) determining cycle number of appearance of a mecA polynucleotide sequence amplified from said sample; (b) determining cycle number of appearance of a bridging region polynucleotide sequence amplified from said sample; and (c) determining cycle number of appearance of an S. aureus-specific polynucleotide sequence amplified from said sample, wherein said S. aureus-specific polynucleotide sequence is not an orfX polynucleotide sequence, (d) comparing said cycle numbers of appearance of said mecA, bridging region, and S. aureus-specific polynucleotide sequences to each other, wherein if said cycle number of appearance of each of said mecA polynucleotide, said bridging region polynucleotide, and said S. aureus-specific polynucleotide sequences are substantially the same, then MRSA is present in said sample. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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28. 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 primers complementary to at least a portion of a bridging sequence; (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 an orfX polynucleotide; and at least one member selected from;
a DNA polymerase enzyme, dNTPs, magnesium and a stabilizer.
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29. A method of detecting an antibiotic-resistant bacterial strain in a sample, wherein said sample contains one or a mixture of bacterial species, said method comprising:
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(a) providing a first set of primers, wherein said first set of primers are capable of producing a first amplification product from at least a portion of a gene, wherein said gene confers antibiotic-resistance; (b) providing a second set of primers, wherein said second set of primers are capable of producing a second amplification product from at least a portion of a bridging region; (c) providing a third set of primers, wherein said third set of primers are capable of producing a third amplification product from at least a portion of a bacterial strain-specific polynucleotide sequence; (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 first, second and third amplification products, (g) comparing said cycle numbers of appearance of said first, second and third amplification products to each other, wherein if said cycle numbers of appearance of said first, second and third amplification products are substantially the same, then an antibiotic-resistant bacterial strain is present in said sample. - View Dependent Claims (30, 31, 32, 33)
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Specification