Method for detecting Chikungunya virus
Compositions, methods and kits for detecting Chikungunya virus (CHIKV) nucleic acids are for detecting very low levels of the viral nucleic acids using nucleic acid amplification. The kit includes a first primer up to 100 bases long and includes a target-complementary 3′ terminal sequence of 15-48 contiguous bases. The first primer optionally may include a first primer 5′ sequence (i.e., an upstream sequence) that is not complementary to CHIKV nucleic acids.
1. A method for determining whether a Chikungunya virus (CHIKV) nucleic acid sequence is present in a test sample comprising nucleic acids, said method comprising:
(a) contacting the nucleic acids of the test sample with a paired set of amplification primers, wherein
i) a first primer of the pair comprises a target-complementary 3′ terminal sequence complementary to at least 20 contiguous bases of nucleotides 35-74 of SEQ ID NO. 4, and
ii) a second primer of the pair comprises a target-complementary sequence complementary to an extension product of the first primer,
(b) performing an in vitro amplification reaction using the nucleic acids of the test sample as templates together with the set of primers, whereby, when the test sample comprises the CHIKV nucleic acid sequence, an amplification product is produced; and
(c) detecting any of the amplification product that is produced in the in vitro amplification reaction,
wherein said detecting of the amplification product in an amount greater than a cutoff value indicates that the CHIKV nucleic acid sequence is present in the test sample, and
wherein said detecting of the amplification product in an amount less than the cutoff value indicates that the CHIKV nucleic acid sequence is absent in the test sample.
2. The method of claim 1 , wherein either of the amplification primers optionally comprises a 5′ sequence that is not complementary to CHIKV.
3. The method of claim 1 , wherein the first primer includes a promoter sequence at its 5′ end.
4. The method of claim 3 , wherein the promotor sequence comprises SEQ ID NO. 90.
5. The method of claim 1 , wherein the target-complementary 3′ terminal sequence of the first primer is comprised in SEQ ID NO. 35.
6. The method of claim 1 , wherein a hybridization probe is used for the detection.
7. The method of claim 6 , wherein the hybridization probe comprises a target-complementary sequence in the range from 10 to 100 nucleotides.
8. The method of claim 7 , wherein the target-complementary sequence of the hybridization probe is comprised in SEQ ID NO. 74.
9. The method of claim 1 , wherein previous to step a), nucleic acids of the test sample are hybridized and captured with an oligonucleotide having a target-complementary sequence comprised in SEQ ID NO. 89 or in SEQ ID NO. 191.
10. The method of claim 1 , wherein a hybridization probe is used for detecting the amplification product, and the hybridization probe comprises a sequence of at least 10 nucleotides that are complementary to SEQ ID NO. 74.
11. The method of claim 1 , wherein the first primer comprises a 5′ terminal sequence that is not complementary to the CHKV nucleic acid sequence.
12. The method of claim 1 , wherein the first primer comprises a promoter primer sequence at its 5′ end, and the promoter primer sequence comprises SEQ ID NO. 115.
13. The method of claim 1 , wherein the target-complementary 3′ terminal sequence comprised in the first primer is complementary to at least 22 contiguous bases of nucleotides 35-74 of SEQ ID NO. 4.
14. The method of claim 1 , wherein the target-complementary 3′ terminal sequence comprised in the first primer is complementary to at least 25 contiguous bases of nucleotides 35-74 of SEQ ID NO. 4.
15. A kit for amplifying and detecting the Chikungunya virus (CHIKV) nucleic acid sequence in the method according to claim 1 , comprising:
i) the first primer comprising the target-complementary 3′ terminal sequence complementary to at least 20 contiguous bases of nucleotides 35-74 of SEQ ID NO. 4, and
ii) the second primer comprising the target-complementary terminal sequence complementary to the extension product of the first primer when SEQ ID NO. 4 is a template in a template-dependent primer extension reaction,
wherein the first primer further comprises a 5′ terminal sequence that is not complementary to the CHKV nucleic acid sequence.
16. The kit of claim 15 , further comprising at least one of:
(i) a hybridization probe comprising a target-complementary sequence in a range from 10 to 100 nucleotides comprised in SEQ ID NO. 74,
(ii) a capture oligonucleotide comprising a target-complementary sequence comprised in SEQ ID NO. 89 or in SEQ ID NO. 191.
17. An amplification primer comprising a target-complementary 3′ terminal sequence complementary to at least 20 contiguous bases of nucleotides 35-74 of SEQ ID NO. 4 and a 5′ sequence that is not complementary to CHIKV.
18. The amplification primer of claim 17 , further comprising a promoter sequence at its 5′ end.
19. The amplification primer of claim 18 , wherein the promotor sequence comprises SEQ ID NO. 90.
20. The amplification primer of claim 17 , wherein the target-complementary 3′ terminal sequence is comprised in SEQ ID NO. 35.