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.

What is claimed is: 
     
         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 an  S. aureus -specific polynucleotide sequence, and wherein said  S. aureus -specific polynucleotide sequence is not an orfX polynucleotide;   (c) combining said first, second set of primers with said sample in a reaction mixture;   (d) performing a multi-cycle amplification reaction with said reaction mixture;   (e) determining cycle numbers of appearance of each of said mecA and  S. aureus -specific polynucleotide sequences,   (f) comparing said cycle numbers of appearance of said mecA 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 said  S. aureus -specific polynucleotide sequence, then MRSA is present in said sample. 
     
     
         2 . A method according to  claim 1 , wherein said first set of primers comprises at least one of SEQ ID NOs: 10-11. 
     
     
         3 . A method according to  claim 1 , wherein said second set of primers comprises at least one of SEQ ID NOs: 13-14 and 18-19. 
     
     
         4 . A method according to  claim 1 , wherein said multi-cycle amplification reaction is a real-time polymerase chain reaction. 
     
     
         5 . A method according to  claim 1 , wherein said  S. aureus -specific polynucleotide sequence is at least a portion of a gene, which is a member selected from nuc and Sa442. 
     
     
         6 . A kit for identifying MRSA in a sample, said kit comprising:
 (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 an  S. aureus -specific polynucleotide sequence, wherein said  S. aureus -specific polynucleotide sequence is not an orfX polynucleotide;   (c) at least one member selected from: a DNA polymerase enzyme, dNTPs, magnesium, and a stabilizer.