Methods of detecting and typing pathogenic strains of francisella tularensis

Abstract

A method of detecting a presence of Francisella tularensis ( F. tularensis ). The method includes amplifying a first nucleic acid from said specimen using a first plurality of primers, said first plurality of primers comprising SEQ ID NO 4 and SEQ ID NO 5. When the first nucleic acid is detected, then the presence of F. tularensis is determined.

1 . A method of detecting a presence of  Francisella tularensis  ( F. tularensis ) or a  F. tularensis  nucleic acid in a specimen, the method comprising:
 amplifying a first nucleic acid from said specimen using a first plurality of primers, said first plurality of primers comprising SEQ ID NO 4 and SEQ ID NO 5; and   detecting the first nucleic acid;   wherein said detecting indicates the presence of  F. tularensis  of  F. tularensis  nucleic acid in the specimen.   
     
     
         2 . The method of  claim 1 , wherein the method includes a singleplex PCR reaction. 
     
     
         3 . The method of  claim 1 , wherein the method includes a multiplex PCR reaction and detecting the first nucleic acid further comprises:
 using a first probe comprising SEQ ID NO 6 for detecting the first nucleic acid.   
     
     
         4 . The method of  claim 1 , wherein the first probe further comprises:
 a fluorescent reporter.   
     
     
         5 . The method of  claim 4 , wherein the first probe further comprises:
 a quencher.   
     
     
         6 . The method of  claim 1 , wherein each primer of the first plurality further comprises a fluorescent reporter. 
     
     
         7 . The method of  claim 1 , further comprising:
 using an endogenous internal control, said using comprising:
 amplifying a second nucleic acid from said specimen using a second plurality of primers, said second plurality of primers comprising SEQ ID NO 1 and SEQ ID NO 2; and 
 detecting the second nucleic acid; 
 wherein said detecting indicates the presence of bacteria or bacterial nucleic acid in the specimen. 
   
     
     
         8 . The method of  claim 7 , wherein detecting the second nucleic acid further comprises:
 using a second probe comprising SEQ ID NO 3.   
     
     
         9 . A detection assay kit for detecting a presence of  Francisella tularensis  ( F. tularensis ) or  F. tularensis  nucleic acid, the kit comprising:
 a first plurality of primers comprising SEQ ID NO 4 and SEQ ID NO 5.   
     
     
         10 . The detection assay kit of  claim 9 , further comprising:
 a first probe comprising a fluorescent reporter dye coupled to an initial 5′-nucleotide of SEQ ID NO 6.   
     
     
         11 . The detection assay kit of  claim 10 , wherein the first probe further comprises:
 a quencher coupled to the initial 3′-nucleotide of SEQ ID NO 6.   
     
     
         12 . The detection assay kit of  claim 10 , further comprising:
 a control assay comprising:
 a second plurality of primers comprising SEQ ID NO 1 and SEQ ID NO 2. 
   
     
     
         13 . The detection assay kit of  claim 12 , further comprising:
 a second probe comprising a fluorescent reporter dye coupled to an initial 5′-nucleotide of SEQ ID NO 3.   
     
     
         14 . The detection assay kit of  claim 10 , further comprising:
 reagents for a PCR process.   
     
     
         15 . The detection assay kit of  claim 9 , wherein the second probe further comprises a quencher coupled to the initial 3′-nucleotide of SEQ ID NO 3. 
     
     
         16 . The detection assay kit of  claim 10 , further comprising:
 reagents for a real time PCR process.   
     
     
         17 . A method of detecting a presence of a virulent strain of  Francisella tularensis  ( F. tularensis ) or nucleic acid of a virulent  F. tularensis  strain in a specimen, the method comprising:
 amplifying a first nucleic acid from said specimen using a first plurality of primers, said first plurality of primers comprising SEQ ID NO 7, and SEQ ID NO 8; and   detecting the first nucleic acid using a first probe comprising SEQ ID NO 9;   wherein said detecting indicates the presence of a virulent strain of  F. tularensis  or nucleic acid of a virulent  F. tularensis  strain in the specimen.   
     
     
         18 . The method of  claim 17 , further comprising:
 amplifying a second nucleic acid from said specimen using a second plurality of primers, said second plurality of primers comprising SEQ ID NO 4 and SEQ ID NO 5; and   detecting the second nucleic acid using a second probe comprising SEQ ID NO 6;   wherein said detecting indicates the presence of any  F. tularensis  strain or nucleic acid of a virulent  F. tularensis  strain in the specimen.   
     
     
         19 . The method of  claim 17 , further comprising:
 using an endogenous internal control, said using comprising:
 amplifying a third nucleic acid from said specimen using a third plurality of primers, said third plurality of primers comprising SEQ ID NO 1 and SEQ ID NO 2; and 
 detecting the third nucleic acid using a third probe comprising SEQ ID NO 3; 
 wherein said detecting indicates the presence of bacteria or bacterial nucleic acid in the specimen. 
   
     
     
         20 . The method of  claim 17 , wherein each primer of the first plurality further comprises a fluorescent reporter. 
     
     
         21 . The method of  claim 17 , wherein the first probe further comprises:
 a fluorescent reporter.   
     
     
         22 . The method of  claim 21 , wherein the first probe further comprises:
 a quencher.   
     
     
         23 - 92 . (canceled)