Oligonucleotide for detecting Salmonella and method of detecting Salmonella

Abstract

The present invention provides oligonucleotides for detecting Salmonella toxin gene invA mRNA and stn mRNA which oligonucleotides specifically bind to invA mRNA or stn mRNA at a relatively low temperature (for example, 41° C.) and at a constant temperature, and a process of amplifying Salmonella toxin gene invA mRNA or stn mRNA and a method of detecting the same using the oligonucleotides.

1 - 3 . (canceled)  
     
     
         4 . A process of amplifying  Salmonella  gene stn mRNA having a specific sequence, comprising 
 obtaining a sample comprising  Salmonella  gene stn mRNA    synthesizing cDNA employing an RNA-dependent DNA polymerase resulting in an RNA/DNA hybrid,    digesting the RNA of the RNA/DNA hybrid with Ribonuclease H to produce a single-stranded DNA,    producing a double-stranded DNA having a promoter sequence capable of transcribing RNA comprising said specific sequence or a sequence complementary to said specific sequence employing a DNA-dependent DNA polymerase, wherein said single-stranded DNA is the template for said producing and wherein said double-stranded DNA produces an RNA transcription product in the presence of an RNA polymerase, and    synthesizing cDNA comprising annealing an oligonucleotide primer pair consisting of (a) a sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and (b) a sequence selected from the group consisting of SEQ ID NOs: 24 to 27 to said RNA transcription product and amplifying said cDNA by employing said RNA-dependent DNA polymerase, where either primer includes an RNA polymerase promoter sequence at the 5′ end.    
     
     
         5 . The process according to  claim 4 , which is a detection method, wherein said amplifying is performed in the presence of an oligonucleotide probe which has a sequence that is complementary to at least a portion of the RNA transcription product, is labeled with an intercalator fluorescent pigment, and has a sequence different from said oligonucleotide primer pair, wherein changes in the fluorescent properties of the intercalator are measured.  
     
     
         6 . The detection method according to  claim 5 , wherein said probe is designed so as to complementarily bind to at least a portion of the sequence of said RNA transcription product, and the fluorescent property changes relative to that of a situation where a complex formation is absent.  
     
     
         7 . (canceled)  
     
     
         8 . The detection method according to  claim 6 , wherein said probe for detecting said stn mRNA comprises at least 10 contiguous bases of SEQ. ID. No. 29 or its complementary sequence.  
     
     
         9 . The process of  claim 4 , wherein said annealing is at a temperature ranging from 35 to 50° C.  
     
     
         10 . The process of  claim 4 , wherein said amplifying said cDNA is at a temperature ranging from 35 to 50° C.  
     
     
         11 . The process of  claim 10 , wherein said amplifying said cDNA is at a constant temperature.  
     
     
         12 . The process of  claim 4 , wherein the process is performed by employing a single enzyme having RNA-dependent DNA polymerase activity, DNA-dependent DNA-polymerase activity, and ribonuclease H activity.  
     
     
         13 . The process of  claim 12 , wherein said enzyme is AMV reverse transcriptase.  
     
     
         14 . The process of  claim 4 , wherein said RNA polymerase is a T7 phage RNA polymerase or a SP6 phage RNA polymerase.  
     
     
         15 . The process of  claim 4 , wherein said sequence selected from the group consisting of SEQ ID NOs: 13 to 18 is the sequence of SEQ ID NO: 15.  
     
     
         16 . The process of  claim 4 , wherein said sequence selected from the group consisting of SEQ ID NOs: 24 to 27 is the sequence of SEQ ID NO: 25.  
     
     
         17 . The detection method according to  claim 5 , wherein said intercalator fluorescent pigment is bonded to a phosphorus atom in the oligonucleotide probe through a linker.  
     
     
         18 . The detection method according to  claim 5 , wherein said oligonucleotide probe is modified at the 3′ hydroxyl group such that extension from said probe is inhibited.  
     
     
         19 . The detection method according to  claim 18 , wherein said oligonucleotide probe is modified at the 3′ hydroxyl group by addition of a glycolic acid.  
     
     
         20 . The detection method according to  claim 5 , wherein said sequence selected from the group consisting of SEQ ID NOs: 13 to 18 is the sequence of SEQ ID NO: 15.  
     
     
         21 . detection method according to  claim 5 , wherein said sequence selected from the group consisting of SEQ ID NOs: 24 to 27 is the sequence of SEQ ID NO: 25.  
     
     
         22 . The detection method according to  claim 8 , wherein said oligonucleotide probe is modified at the 3′ hydroxyl group such that extension from said probe is inhibited.  
     
     
         23 . The detection method according to  claim 22 , wherein said oligonucleotide probe is modified at the 3′ hydroxyl group by addition of a glycolic acid.