Method for detecting the presence of a hypervirulent
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. A method of detecting the presence of a hypervirulent Clostridium difficile strain in a biological sample, the method comprising:
performing a nucleic acid amplification reaction comprising DNA extracted from the biological sample as a template, a first oligonucleotide primer set specific for amplifying a target sequence in the C. difficile hydR gene in the reaction, wherein said hydR gene target sequence comprises at least part of the sequence set forth in SEQ ID NO: 1, and a second oligonucleotide primer set specific for amplifying at least part of the target sequence corresponding to C. difficile sequence set forth in SEQ ID NO:2 in the reaction.
2. The method according to claim 1 , further comprising a step of detecting the presence of a hypervirulent Clostridium difficile strain in said biological sample, wherein the hypervirulent Clostridium difficile strain is detected in the sample, when the first oligonucleotide primer set does not amplify a specific product and the second oligonucleotide primer set amplifies a specific product.
3. The method according to claim 1 , wherein the hypervirulent Clostridium difficile strain is Clostridium difficile strain 027 or a 027-ribotype-resembling Clostridium difficile strain.
4. The method according to claim 1 , wherein the presence of C. difficile hydR gene DNA in said sample indicates that Clostridium difficile strain 027 is not present in the sample.
5. The method according to claim 1 , wherein the C. difficile -specific target sequence for the first oligonucleotide primer set is a nucleotide region of an C. difficile hydR gene as set forth in SEQ ID NO: 1 and at least part of said nucleotide region is specifically amplified.
6. The method according to claim 5 , wherein the first oligonucleotide primer set comprises an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO: 3 and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO: 4.
7. The method according to claim 6 , wherein the first oligonucleotide primer set comprises an oligonucleotide comprising or consisting of the nucleotide sequence as set forth in SEQ ID NO: 3 and an oligonucleotide comprising or consisting of the nucleotide sequence as set forth in SEQ ID NO: 4.
8. The method according to claim 1 , wherein the presence of the target sequence amplified with the first oligonucleotide primer set is detected by the use of a probe comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:7.
9. The method according to claim 8 , wherein the presence of the target sequence amplified with the first oligonucleotide primer set is detected by the use of a probe comprising or consisting of the nucleotide sequence as set forth in SEQ ID NO:7.
10. The method according to claim 1 , wherein the second oligonucleotide primer set comprises an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 5 and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 6.
11. The method according to claim 10 , wherein the second oligonucleotide primer set comprises an oligonucleotide comprising or consisting of the nucleotide sequence as set forth in SEQ ID NO: 5 and an oligonucleotide comprising or consisting of the nucleotide sequence as set forth in SEQ ID NO: 6.
12. The method according to claim 1 , wherein the presence of the target sequence amplified with the second oligonucleotide primer set is detected by the use of a probe comprising or consisting of the nucleotide sequence as set forth in SEQ ID NO:8 or 9.
13. The method according to claim 1 , wherein the amplification reaction further comprises a third oligonucleotide primer set specific for amplifying C. difficile toxin B gene (tcdB) in the reaction and at least part of nucleotide region as set forth in SEQ ID NO: 10 is specifically amplified in the reaction.
14. The method according to claim 13 , wherein the third oligonucleotide primer set comprises an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO: 11 and an oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO: 12.
15. The method according to claim 14 , wherein the third oligonucleotide primer set comprises an oligonucleotide comprising or consisting of the nucleotide sequence as set forth in SEQ ID NO: 11 and an oligonucleotide comprising or consisting of the nucleotide sequence as set forth in SEQ ID NO: 12.
16. The method according to claim 13 , wherein the presence of the target sequence amplified with the third oligonucleotide primer set is detected by the use of a probe comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO:13.
17. The method according to claim 16 , wherein the presence of the target sequence amplified with the third oligonucleotide primer set is detected by the use of a probe comprising or consisting of the nucleotide sequence as set forth in SEQ ID NO: 13.
18. The method according to claim 1 , wherein the biological sample is a stool sample or a food sample.
19. The method according to claim 2 , wherein the detection of hypervirulent Clostridium difficile strain is performed using a DNA chip, gel electrophoresis, a radiation measurement, a fluorescence measurement, or a phosphorescence measurement.
20. The method according to claim 1 , wherein the method is performed as a real-time PCR assay.
21. The method according to claim 1 , wherein the nucleic acid amplification reaction is a PCR reaction.
22. The method according to claim 21 , wherein the PCR reaction is performed as a real-time PCR assay.
23. The method according to claim 1 , wherein the first oligonucleotide primer set amplifies at least 50 contiguous nucleotides of SEQ ID NO:1.
24. The method according to claim 1 , wherein the second oligonucleotide primer set amplifies at least 50 contiguous nucleotides of SEQ ID NO:2.
25. The method according to claim 6 , wherein the oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO: 3 and the oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in the nucleotide sequence as set forth in SEQ ID NO: 4 are each less than 30 nucleotides in length.
26. The method according to claim 6 , wherein at least one oligonucleotide of the first oligonucleotide primer set comprises a modification selected from the group consisting of a base modification, a sugar modification, and a backbone modification.
27. The method according to claim 26 , wherein at least one oligonucleotide of the first oligonucleotide primer set comprises a modified nucleotide selected from the group consisting of a LNA nucleotide, a minor groove binder (MGB)-conjugated nucleotide, a nucleotide with a SuperBase, and a PNA nucleotide.
28. The method according to claim 10 , wherein the oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 5 and the oligonucleotide comprising or consisting of at least 10 contiguous nucleotides present in a nucleotide sequence as set forth in SEQ ID NO: 6 are each less than 30 nucleotides in length.
29. The method according to claim 10 , wherein at least one oligonucleotide of the second oligonucleotide primer set comprises a modification selected from the group consisting of a base modification, a sugar modification, and a backbone modification.
30. The method according to claim 29 , wherein at least one oligonucleotide of the first oligonucleotide primer set comprises a modified nucleotide selected from the group consisting of a LNA nucleotide, a minor groove binder (MGB)-conjugated nucleotide, a nucleotide with a SuperBase, and a PNA nucleotide.