Compositions and methods for the rapid differential detection of Zika virus
This invention relates to compositions and methods for the differential detection of multiple viruses using a one-step assay. The viruses to be detected include Zika, West Nile, dengue (genotype 1-4) and chikungunya viruses. In particular, the invention relates to a method of and assay for differential detection of the viruses using specific primers and probes designed to detect and differentiate between the viruses.
1. A method of differentially detecting the presence of the Zika virus, dengue virus (genotype 1-4), chikungunya virus, and West Nile virus, in a sample comprising:
contacting the sample with at least one primer specific the Zika virus and at least one primer specific for each of dengue virus (genotype 1-4), chikungunya virus, and West Nile virus, wherein the primers allow the differential detection of Zika virus from dengue virus (genotype 1-4), chikungunya virus, and West Nile virus;
subjecting the sample and the primers to amplification conditions;
detecting the presence of amplification product, wherein the presence of amplification product from the primer specific to Zika virus indicates the presence of nucleic acid from Zika virus in the sample, and the presence of amplification product from the primer specific for dengue virus (genotype 1-4), Chikungunya virus, and West Nile virus indicates the presence of nucleic acid from that virus in the sample.
2. The method of claim 1 , further comprising contacting the sample with a probe specific for Zika virus, dengue virus (genotype 1-4), chikungunya virus, and West Nile virus.
3. The method of claim 1 , wherein the sample is of natural origin and is chosen from the group consisting of plasma, serum, whole blood, spinal fluid, semen, amniotic fluid, lymph fluid, synovial fluid, urine, tears, blood cells, organs, and tissue.
4. The method of claim 1 , wherein the sample is urine or serum.
5. The method of claim 1 , wherein the sample is from a human subject.
6. The method of claim 1 , wherein the nucleic acid is RNA.
7. The method of claim 2 , wherein the primers and probe specific for Zika virus are from the 3′UTR of the viral genome.
8. The method of claim 7 , wherein the primers comprise SEQ ID NOs: 1 and 2, and the probe comprises SEQ ID NO: 3.
9. A method of detecting the presence of the Zika virus and at least one other virus chosen from the group consisting of dengue virus (genotype 1-4), chikungunya virus, and West Nile virus, in a sample comprising:
contacting the sample with primers specific the Zika virus comprising SEQ ID NOs: 1 and 2, and at least one primer specific from another virus chosen from the group consisting of dengue virus (genotype 1-4), chikungunya virus, and West Nile virus;
subjecting the sample and the primers to amplification conditions;
further contacting the sample with a probe comprising SEQ ID NO: 3 and wherein the probe comprises a 5′ modification of CAL Fluor Red 610 and a 3′ modification of BHQ-2; and
detecting the presence of amplification product, wherein the presence of amplification product from the primer specific to Zika virus indicates the presence of nucleic acid from Zika virus in the sample, and the presence of amplification product from the primer specific for the at least one virus chosen from the group consisting of dengue virus (genotype 1-4), Chikungunya virus, and West Nile virus indicates the presence of nucleic acid from that virus in the sample.
10. The method of claim 2 , wherein the primers and probe specific for dengue virus (genotype 1-4) are from the 3′UTR of the viral genome.
11. The method of claim 6 , wherein the primers comprise SEQ ID NOs: 7, 8, and 9, and the probe comprises SEQ ID NO: 10.
12. The method of claim 7 , wherein the probe comprises a 5′ modification of CAL Fluor Orange 560 and a 3′ modification of BHQ-1Plus.
13. The method of claim 2 , wherein the primers and probe specific for Chikungunya virus are from the NSP2 portion of the viral genome.
14. The method of claim 9 , wherein the primers comprise SEQ ID NOs: 11 and 12, and the probe comprises SEQ ID NO: 13.
15. The method of claim 10 , wherein the probe comprises a 5′ modification of FAM and a 3′ modification of BHQ-1Plus.
16. The method of claim 2 , wherein the primers and probe specific for West Nile virus are from the NS5 portion of the viral genome.
17. The method of claim 16 , wherein the primers comprise SEQ ID NOs: 4 and 5, and the probe comprise SEQ ID NO: 6.
18. The method of claim 17 , wherein the probe comprises a 5′ modification of Quasar 670 and a 3′ modification of BHQ-2.
19. A kit for the simultaneous detection and differentiation of Zika virus and at least one other virus chosen from the group consisting of dengue virus (genotype 1-4), chikungunya virus, and West Nile virus, comprising: primers comprising SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 11, and 12; and probes comprising SEQ ID NOs: 3, 6, 10, and 13.
20. The kit of claim 19 , further comprising reagents for performing the differential detection comprising control sequences, nucleic acid polymerase and nucleic acid extraction reagents; and instructions for use.
21. The kit of claim 19 , wherein the primers and probes for the detection of each virus are in a single reaction mixture.
22. The kit of claim 19 , wherein the probes further comprise a detectable label.
23. A synthetic nucleic acid with a nucleic acid sequence selected from any of one of SEQ ID NOs: 1-13.
24. The method of claim 7 , wherein the primers consist of SEQ ID NOs: 1 and 2, and the probe consists of SEQ ID NO: 3.