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

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.

1 . A method of determining whether a sample contains a single bacterial species or a mixture of different bacterial species, said method comprising:
 (a) providing a first set of primers, wherein said first set of primers are capable of amplifying at least a portion of a gene that confers antibiotic resistance;   (b) providing a second set of primers, wherein said second set of primers are capable of amplifying at least a portion of a first bacterial strain-specific polynucleotide sequence;   (c) providing a third set of primers, wherein said third set of primers are capable of amplifying at least a portion of a second bacterial strain-specific polynucleotide sequence;   (c) combining said first, second and third set of primers with said sample to produce a reaction mixture;   (d) performing a multi-cycle amplification reaction with said reaction mixture to produce a first amplification product with said first set of primers, a second amplification product with said second set of primers, and a third amplification product with said third set of primers;   (e) determining cycle numbers of appearance of said first, second and third amplification products,   wherein if said cycle numbers of appearance of said first, second and third amplification products are substantially the same, then said sample contains a single bacterial species, and wherein if said cycle numbers of appearance are not substantially the same, then said sample contains a mixture of a strain of antibiotic-sensitive bacteria and a different strain of antibiotic-resistant bacteria.   
     
     
         2 . The method of  claim 1 , wherein said gene confers resistance to methicillin. 
     
     
         3 . The method of  claim 1 , wherein said gene confers resistance to vancomycin. 
     
     
         4 . A method of determining whether a sample contains a single bacterial strain or species or a mixture of different bacterial strains or species, said method comprising:
 (a) providing a first set of primers, wherein said first set of primers are capable of amplifying at least a portion of a first locus of a bacterial genome;   (b) providing a second set of primers, wherein said second set of primers are capable of amplifying at least a portion of a second locus of a bacterial genome, wherein said second locus is a strain-specific locus;   (c) providing a third set of primers, wherein said third set of primers are capable of amplifying at least a portion of a third locus of a bacterial genome, wherein said third locus is a strain-specific locus;   (c) combining said first, second and third set of primers with said sample to produce a reaction mixture;   (d) performing a multi-cycle amplification reaction with said reaction mixture to produce a first amplification product with said first set of primers, a second amplification product with said second set of primers, and a third amplification product with said third set of primers;   (e) determining cycle numbers of appearance of said first, second and third amplification products,   wherein if said cycle numbers of appearance of said first, second and third amplification products are substantially the same, then said sample contains a single bacterial strain or species, and wherein if said cycle numbers of appearance are not substantially the same, then said sample contains a mixture of a bacterial strains or species.