COMPOSITIONS AND KITS FOR DETECTING THE PRESENCE OF A HYPERVIRULENT CLOSTRIDIUM DIFFICILE STRAIN
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 . An oligonucleotide primer set comprising a first oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:5 and a second oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:6, wherein the primer set amplifies a target sequence in the C. difficile genome, and wherein the primer set further comprises a probe for detecting the amplified target sequence, wherein the probe comprises one or more modified nucleotides and/or a fluorescent label, radiolabel, or phosphorescent label.
2 . The oligonucleotide primer set according to claim 1 , wherein the first oligonucleotide comprises the nucleotide sequence as set forth in SEQ ID NO:5 and the second oligonucleotide comprises the nucleotide sequence as set forth in SEQ ID NO:6.
3 . The oligonucleotide primer set according to claim 2 , wherein the first oligonucleotide consists of the nucleotide sequence as set forth in SEQ ID NO:5 and the second oligonucleotide consists of the nucleotide sequence as set forth in SEQ ID NO:6.
4 . The oligonucleotide primer set according to claim 1 , wherein the probe comprises at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:8 or SEQ ID NO:9.
5 . The oligonucleotide primer set according to claim 4 , wherein the probe comprises the nucleotide sequence as set forth in SEQ ID NO:8 or SEQ ID NO:9.
6 . The oligonucleotide primer set according to claim 1 , further comprising a second oligonucleotide primer set, wherein the second oligonucleotide primer set comprises a third oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:3 and a fourth oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:4, wherein the second oligonucleotide primer set amplifies a target sequence in the C. difficile hydR gene.
7 . The oligonucleotide primer set according to claim 6 , wherein the third oligonucleotide comprises or consists of the nucleotide sequence as set forth in SEQ ID NO:3 and the fourth oligonucleotide comprises or consists of the nucleotide sequence as set forth in SEQ ID NO:4.
8 . The oligonucleotide primer set according to claim 6 , further comprising a probe for detecting the amplified hydR target sequence, said probe comprising at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:7.
9 . The oligonucleotide primer set according to claim 8 , wherein the probe for detecting the amplified hydR target sequence comprises the nucleotide sequence as set forth in SEQ ID NO:7.
10 . The oligonucleotide primer set according to claim 6 , further comprising a third oligonucleotide primer set, wherein the third oligonucleotide primer set comprises a fifth oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:11 and a sixth oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:12, wherein the third oligonucleotide primer set amplifies a target sequence in the C. difficile tcdB gene.
11 . The oligonucleotide primer set according to claim 10 , wherein the fifth oligonucleotide comprises or consists of the nucleotide sequence as set forth in SEQ ID NO:11 and the sixth oligonucleotide comprises or consists of the nucleotide sequence as set forth in SEQ ID NO:12.
12 . The oligonucleotide primer set according to claim 10 , further comprising a probe for detecting the amplified tcdB target sequence, said probe comprising at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:13.
13 . The oligonucleotide primer set according to claim 12 , wherein the probe for detecting the amplified tedB target sequence comprises the nucleotide sequence as set forth in SEQ ID NO:13.
14 . A kit for detecting a hypervirulent C. difficile strain in a biological sample, the kit comprising:
(i) an oligonucleotide primer set comprising a first oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:5 and a second oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:6, wherein the primer set amplifies a target sequence in the C. difficile genome;
(ii) a probe for detecting the amplified target sequence, wherein the probe comprises one or more modified nucleotides and/or a fluorescent label, radiolabel, or phosphorescent label; and
(iii) a reagent for performing amplification of a nucleic acid.
15 . The kit of claim 14 , wherein the reagent is selected from the group consisting of a DNA polymerase, dNTPs, and a buffer.
16 . The kit of claim 14 , wherein the first oligonucleotide comprises or consists of the nucleotide sequence as set forth in SEQ ID NO:5 and the second oligonucleotide comprises or consists of the nucleotide sequence as set forth in SEQ ID NO:6.
17 . The kit of claim 14 , wherein the probe comprises at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:8 or SEQ ID NO:9.
18 . The kit of claim 14 , further comprising a second oligonucleotide primer set, wherein the second oligonucleotide primer set comprises a third oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:3 and a fourth oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:4, wherein the second oligonucleotide primer set amplifies a target sequence in the C. difficile hydR gene.
19 . The kit of claim 18 , wherein the third oligonucleotide comprises or consists of the nucleotide sequence as set forth in SEQ ID NO:3 and the fourth oligonucleotide comprises or consists of the nucleotide sequence as set forth in SEQ ID NO:4.
20 . The kit of claim 18 , further comprising a third oligonucleotide primer set, wherein the third oligonucleotide primer set comprises a fifth oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:11 and a sixth oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:12, wherein the third oligonucleotide primer set amplifies a target sequence in the C. difficile tcdB gene.
21 . The kit of claim 20 , wherein the fifth oligonucleotide comprises or consists of the nucleotide sequence as set forth in SEQ ID NO:11 and the sixth oligonucleotide comprises or consists of the nucleotide sequence as set forth in SEQ ID NO: 12.