Primers for use in detecting beta-lactamases

Abstract

Oliognucleotide primers are provided that are specific for nucleic acid characteristic of certain beta-lactamases. The primers can be employed in methods to identify nucleic acid characteristic of family-specific beta-lactamase enzymes in samples, and particularly, in clinical isolates of Gram-negative bacteria.

1 - 89 . (canceled) 
     
     
         90 . A primer selected from the group of:
 5′-CTT GGT CTG ACA GTT ACC-3′ (SEQ ID NO: 3);   5′-TGT CGC CCT TAT TCC-3′ (SEQ ID NO: 4); and   5′-TCG GGG AAA TGT GCG-3′ (SEQ ID NO:5);   and full length complements thereof.   
     
     
         91 . A method for identifying a beta-lactamase in a clinical sample, the method comprising:
 providing a pair of oligonucleotide primers specific for nucleic acid characteristics of the TEM family of beta-lactamase enzymes, wherein one primer of the pair is complementary to at least a portion of the beta-lactamase nucleic acid in the sense strand and the other primer of each pair is complementary to at least a portion of the beta-lactamase nucleic acid in the antisense strand;   annealing the primers to the beta-lactamase nucleic acid;   simultaneously extending the annealed primers from a 3′ terminus of each primer to synthesize an extension product that is complementary to the nucleic acid strands annealed to each primer wherein each extension product after separation from the beta-lactamase nucleic acid serves as a template for the synthesis of an extension product for the other primer of each pair;   separating the amplified products; and   analyzing the separated amplified products for a region characteristic of the beta-lactamase.   
     
     
         92 . A diagnostic kit for detecting a TEM family beta-lactamase which comprises packaging, containing, separately packaged:
 (a) at least one primer pair capable of hybridizing to beta-lactamase nucleic acid of interest;   (b) a positive and negative control; and   (c) a protocol for identification of the beta-lactamase nucleic acid of interest.   
     
     
         93 . A method for identifying a beta-lactamase enzyme in a clinical sample, the method comprising:
 providing a pair of oligonucleotide primers, wherein at least one of the primers is selected from the primers of  claim 90 , and further wherein one primer of the pair is complementary to at least a portion of the beta-lactamase nucleic acid in the sense strand and the other primer of each pair is complementary to at least a portion of the beta-lactamase nucleic acid in the antisense strand;   annealing the primers to the beta-lactamase nucleic acid;   simultaneously extending the annealed primers from a 3′ terminus of each primer to synthesize an extension product that is complementary to the nucleic acid strands annealed to each primer wherein each extension product after separation from the beta-lactamase nucleic acid serves as a template for the synthesis of an extension product for the other primer of each pair;   separating the amplified products; and   analyzing the separated amplified products for a region characteristic of the beta-lactamase.   
     
     
         94 . The method of  claim 93  wherein the beta-lactamase enzyme is found in a Gram-negative bacterium. 
     
     
         95 . The method of  claim 94  wherein the Gram-negative bacterium is selected from the group consisting of  Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Providenceia  supp.,  Proteus mirabilis, Yersinia enterocolitica , and combinations thereof. 
     
     
         96 . A diagnostic kit for detecting a beta-lactamase enzyme in a clinical sample which comprises packaging, containing, separately packaged:
 (a) a pair of oligonucleotide primers capable of hybridizing to beta-lactamase nucleic acid of interest, wherein at least one of the primers is selected from the primers of  claim 90 ;   (b) a positive and negative control; and   (c) a protocol for identification of the beta-lactamase nucleic acid of interest.   
     
     
         97 . The kit of  claim 96  wherein the beta-lactamase enzyme is found in a Gram-negative bacterium. 
     
     
         98 . The kit of  claim 97  wherein the Gram-negative bacterium is selected from the group consisting of  Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Providenceia  supp.,  Proteus mirabilis, Yersinia enterocolitica , and combinations thereof. 
     
     
         99 . A method for analyzing a beta-lactamase of the TEM family of beta-lactamases in a clinical sample, the method comprising:
 providing a clinical sample suspected of containing a beta-lactamase nucleic acid of the TEM family of beta-lactamases;   providing a pair of oligonucleotide primers, wherein the primers specifically hybridize with a beta-lactamase nucleic acid of the TEM family of beta-lactamases, wherein one primer of the pair is complementary to at least a portion of the sense strand of the beta-lactamase nucleic acid and the other primer of the pair is complementary to at least a portion of the antisense strand of the beta-lactamase nucleic acid, and wherein at least one of the primers is selected from the group consisting of:   5′-CTT GGT CTG ACA GTT ACC-3′ (SEQ ID NO: 3);   5′-TGT CGC CCT TAT TCC-3′ (SEQ ID NO: 4); and   5′-TCG GGG AAA TGT GCG-3′ (SEQ ID NO:5);   and full length complements thereof;   annealing the primers to the beta-lactamase nucleic acid of the TEM family of the beta-lactamases if the beta-lactamase nucleic acid of the TEM family of beta-lactamases is in the sample;   simultaneously extending the annealed primers from a 3′ terminus of each primer of the pair and synthesizing extension products that are complementary to the nucleic acid strands of the beta-lactamase nucleic acid annealed to each primer of the pair wherein the extension products after separation from one strand of the beta-lactamase nucleic acid serve as templates for the synthesis of extension products of other strands of the beta-lactamase nucleic acid;   separating the extension products; and   analyzing the separated extension products for a region characteristic of the beta-lactamase of the TEM family if the beta-lactamase nucleic acid of the TEM family of beta-lactamases is in the sample.   
     
     
         100 . The method of  claim 99  wherein the TEM family of beta-lactamases are found in a Gram-negative bacterium. 
     
     
         101 . The method of  claim 100  wherein the Gram-negative bacterium is selected from the group consisting of  Enterobacter cloacae, Citrobacter, freundii, Serratia marcescens, Providencia  spp.,  Proteus mirabilis , and  Yersinia enterocolitica.    
     
     
         102 . A diagnostic kit for detecting a beta-lactamase in a clinical sample which comprises:
 (a) a pair of oligonucleotide primers capable of hybridizing to a beta-lactamase nucleic acid of interest, wherein the beta-lactamase nucleic acid of interest is a beta-lactamase nucleic acid of the TEM family of beta-lactamases;   (b) a positive and negative control; and   (c) a protocol for identification of the beta-lactamase nucleic acid of interest; wherein at least one of the primers is selected from the group consisting of:   5′-CTT GGT CTG ACA GTT ACC-3′ (SEQ ID NO: 3);   5′-TGT CGC CCT TAT TCC-3′ (SEQ ID NO: 4); and   5′-TCG GGG AAA TGT GCG-3′ (SEQ ID NO:5);   and full length complements thereof.   
     
     
         103 . The kit of  claim 102  wherein the TEM family of beta-lactamases are found in a Gram-negative bacterium. 
     
     
         104 . The kit of  claim 103  wherein the Gram-negative bacterium is selected from the group consisting of  Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Providencia  spp.,  Proteus mirabilis , and  Yersinia enterocolitica.