Method for detecting Chikungunya virus
View Patent ↗Compositions, methods and kits for detecting Chikungunya viral nucleic acids. Particularly described are methods for detecting very low levels of the viral nucleic acids using nucleic acid amplification.
1. A kit for amplifying and detecting a Chikungunya virus (CHIKV) nucleic acid sequence, comprising:
(a) a first primer up to 100 bases long, wherein the 3′ terminal sequence of said first primer consists of SEQ ID NO:108, and wherein said first primer comprises a first primer 5′ phage T7 promoter sequence that is not complementary to CHIKV nucleic acids;
(b) a second primer up to 100 bases long, wherein the 3′ terminal sequence of said second primer is selected from the group consisting of SEQ ID NO:148, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:175 and SEQ ID NO:176, and wherein said second primer optionally comprises a second primer 5′ sequence that is not complementary to CHIKV nucleic acids; and
(c) a hybridization probe for detecting a nucleic acid amplification product synthesized using said primers, wherein said primers and said hybridization probe are in packaged combination with each other.
2. The kit of claim 1 , wherein said hybridization probe is up to 40 bases in length and comprises 15-40 contiguous bases of SEQ ID NO:84.
3. The kit of claim 1 , wherein the target-complementary 3′ terminal sequence of the second primer is selected from the group consisting of SEQ ID NO:148, SEQ ID NO:170, SEQ ID NO:171, and SEQ ID NO:172.
4. The kit of claim 3 , wherein the hybridization probe consists of SEQ ID NO:164.
5. The kit of claim 1 , wherein the target-complementary 3′ terminal sequence of the second primer is selected from the group consisting of SEQ ID NO:174, SEQ ID NO:175 and SEQ ID NO:176.
6. The kit of claim 5 , wherein said hybridization probe is selected from the group consisting of SEQ ID NO:184 and SEQ ID NO:185.
7. The kit of claim 1 , further comprising a third primer up to 100 bases long and comprising a target-complementary 3′ terminal sequence consisting of 15-47 contiguous bases of SEQ ID NO:186, said target-complementary 3′ terminal sequence of said third primer being fully contained within the sequence of SEQ ID NO:186, said third primer optionally comprising a third primer 5′ sequence that is not complementary to CHIKV nucleic acids, and said third primer being different from said second primer.
8. The kit of claim 7 , wherein each of said second and third primers that are different from each other comprise target-complementary 3′ terminal sequences consisting of 15-47 contiguous bases of SEQ ID NO:187.
9. The kit of claim 8 , wherein the target-complementary 3′ terminal sequence of the second primer is SEQ ID NO:148.
10. The kit of claim 9 , wherein the target-complementary 3′ terminal sequence of the third primer is selected from the group consisting of SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173.
11. The kit of claim 7 , wherein each of said second and third primers that are different from each other comprise target-complementary 3′ terminal sequences consisting of 15-40 contiguous bases of SEQ ID NO:84.
12. A method for determining whether a Chikungunya virus (CHIKV) nucleic acid sequence is present in a test sample comprising nucleic acids, said method comprising the steps of:
(a) contacting nucleic acids of the test sample with a set of amplification oligonucleotides that comprises,
a first amplification oligonucleotide, the 3′ terminal sequence of said first amplification oligonucleotide consisting of SEQ ID NO:108, and
a second amplification oligonucleotide selected from the group consisting of SEQ ID NO:148, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:175, and SEQ ID NO:176;
(b) performing an in vitro nucleic acid amplification reaction using nucleic acids of the test sample as templates together with said set of amplification oligonucleotides, whereby, if said test sample comprises said CHIKV nucleic acid sequence, there is produced an amplification product; and
(c) detecting any of said amplification product that may have been produced in the in vitro nucleic acid amplification reaction, wherein detecting said 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 detecting said amplification product in an amount less than the cutoff value indicates that the CHIKV nucleic acid sequence is absent from the test sample.
13. The method of claim 12 , wherein step (c) comprises detecting said amplification product using a hybridization probe.
14. The method of claim 13 , wherein said second amplification oligonucleotide is SEQ ID NO:148, and wherein said hybridization probe is SEQ ID NO:164.
15. The method of claim 13 , wherein said second amplification oligonucleotide is selected from the group consisting of SEQ ID NO:174, SEQ ID NO:175, and SEQ ID NO:176, and wherein said hybridization probe is selected from the group consisting of SEQ ID NO:184 and SEQ ID NO:185.
16. The method of claim 12 , wherein said second amplification oligonucleotide is SEQ ID NO:148, wherein step (c) comprises detecting said amplification product using a hybridization probe, and wherein the probability of detecting said amplification product in the amount greater than the cutoff value is at least 95% when the concentration of the CHIKV nucleic acid sequence in the test sample is in the range of from 26 copies/ml to about 3,400 copies/ml.
17. The method of claim 12 , wherein said second amplification oligonucleotide is SEQ ID NO:148, wherein step (c) comprises detecting said amplification product using a hybridization probe, and wherein the probability of detecting said amplification product in the amount greater than the cutoff value is at least 95% when the concentration of the CHIKV nucleic acid sequence in the test sample is in the range of from 26 copies/ml to about 200 copies/ml.
18. The method of claim 12 , wherein said second amplification oligonucleotide is SEQ ID NO:148, wherein step (c) comprises detecting said amplification product using a hybridization probe, and wherein the probability of detecting said amplification product in the amount greater than the cutoff value is at least 95% when the concentration of the CHIKV nucleic acid sequence in the test sample is in the range of from 100 copies/ml to about 3,400 copies/ml.
19. The method of claim 12 , wherein step (c) comprises detecting said amplification product using a hybridization probe, wherein the probability of detecting said amplification product in the amount greater than the cutoff value is at least 95% when the concentration of the CHIKV nucleic acid sequence in the test sample is in the range of from 26 copies/ml to about 200 copies/ml, and wherein the first amplification oligonucleotide comprises a phage T7 promoter sequence located upstream of SEQ ID NO:108.
20. The kit of claim 1 , further comprising an additional primer up to 100 bases long, wherein the 3′ terminal sequence of said additional primer consists of SEQ ID NO:109, and wherein said additional primer optionally comprises a 5′ sequence that is not complementary to CHIKV nucleic acids.
21. The kit of claim 20 , wherein said additional primer comprises the optional 5′ sequence, and wherein the optional 5′ sequence comprises a phage T7 promoter sequence.
22. The method of claim 12 , wherein the set of amplification oligonucleotides in step (a) further comprises an additional primer up to 100 bases long, wherein the 3′ terminal sequence of said additional primer consists of SEQ ID NO:109, and wherein said additional primer optionally comprises a 5′ sequence that is not complementary to CHIKV nucleic acids.
23. The method of claim 22 , wherein said additional primer comprises the optional 5′ sequence, and wherein the optional 5′ sequence comprises a phage T7 promoter sequence.
24. The method of claim 12 , wherein the first amplification oligonucleotide comprises a phage T7 promoter sequence located upstream of SEQ ID NO:108.
25. The method of claim 24 , wherein the base sequence of the first amplification oligonucleotide is the base sequence of SEQ ID NO:128.
26. The kit of claim 1 , wherein the base sequence of the first primer is SEQ ID NO:128.