Methods for Detecting the Presence of a Hypervirulent Clostridium Difficile Strain

Abstract

The present invention provides a nucleic acid amplification based method for detecting a hypervirulent Clostridium difficile strain in a biological sample. The present invention is based on the use of oligonucleotide primers and probes specific to negative and positive markers in hypervirulent Clostridium difficile genome.

1 .- 20 . (canceled) 
     
     
         21 . An oligonucleotide primer set comprising a first oligonucleotide comprising or consisting of a nucleotide sequence of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 3, a second oligonucleotide comprising or consisting of a nucleotide sequence of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 4, wherein the oligonucleotide primer set amplifies a target sequence in the  C. difficile  hydR gene, and a probe, wherein the probe comprises one or more modified nucleotides and/or a fluorescent label, radiolabel, or phosphorescent label. 
     
     
         22 . The oligonucleotide primer set according to  claim 21 , wherein the first oligonucleotide comprises the nucleotide sequence as set forth in SEQ ID NO: 3 and the second oligonucleotide comprises the nucleotide sequence as set forth in SEQ ID NO: 4. 
     
     
         23 . The oligonucleotide primer set according to  claim 22 , wherein the first oligonucleotide consists of the nucleotide sequence as set forth in SEQ ID NO: 3 and the second oligonucleotide consists of the nucleotide sequence as set forth in SEQ ID NO: 4. 
     
     
         24 . The oligonucleotide primer set according to  claim 21 , further comprising a probe comprising or consisting of a nucleotide sequence of at least 10 contiguous nucleotides as set forth in SEQ ID NO: 7. 
     
     
         25 . The oligonucleotide primer set according to  claim 24 , wherein said probe sequence comprises or consists of the nucleotide sequence as set forth in SEQ ID NO: 7. 
     
     
         26 .- 37 . (canceled) 
     
     
         38 . An oligonucleotide probe comprising an oligonucleotide comprising or consisting of a nucleotide sequence of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 7, wherein the probe comprises one or more modified nucleotides and/or a fluorescent label, radiolabel, or phosphorescent label. 
     
     
         39 .- 40 . (canceled) 
     
     
         41 . A kit for detecting a hypervirulent  Clostridium difficile  strain in a biological sample, the kit comprising: the oligonucleotide primer set according to any one of  claims 21 - 23 ; and a reagent for performing amplification on a nucleotide acid. 
     
     
         42 . The kit according to  claim 41 , wherein the reagent is selected from the group consisting of: DNA polymerase, dNTPs, and a buffer. 
     
     
         43 . An oligonucleotide primer set for generating an amplicon from a  C. difficile  hydR gene, wherein the primer set comprises a first primer comprising a nucleotide sequence consisting of SEQ ID NO: 3, a second primer wherein the second primer is from 10 to 21 contiguous nucleotides in length, and a probe, wherein the probe comprises one or more modified nucleotides and/or a fluorescent label, radiolabel, or phosphorescent label, and wherein the first and second primers are configured to amplify at least 50 nucleotides of SEQ ID NO: 1. 
     
     
         44 . An oligonucleotide primer set for generating an amplicon from a  C. difficile  hydR gene, wherein the primer set comprises a first primer comprising a nucleotide sequence consisting of from 10 to 20 contiguous nucleotides, a second primer comprising a nucleotide sequence consisting of SEQ ID NO: 4, and a probe, wherein the probe comprises one or more modified nucleotides and/or a fluorescent label, radiolabel, or phosphorescent label, and wherein the first and second primers are configured to amplify at least 50 nucleotides of SEQ ID NO: 1. 
     
     
         45 . The oligonucleotide primer set according to  claim 22 , wherein the probe comprises a nucleotide sequence of at least 10 contiguous nucleotides as set forth in SEQ ID NO: 7. 
     
     
         46 . The oligonucleotide primer set according to  claim 23 , wherein the probe comprises a nucleotide sequence of at least 10 contiguous nucleotides as set forth in SEQ ID NO: 7. 
     
     
         47 . The oligonucleotide primer set according to  claim 43 , wherein the probe comprises a nucleotide sequence of at least 10 contiguous nucleotides as set forth in SEQ ID NO: 7. 
     
     
         48 . The oligonucleotide primer set according to  claim 44 , wherein the probe comprises a nucleotide sequence of at least 10 contiguous nucleotides as set forth in SEQ ID NO: 7.