Detection of West Nile virus nucleic acids in the viral 3′ non-coding region
View Patent ↗Methods for detecting flavivirus nucleic acids. Particularly described are methods for detecting West Nile virus nucleic acids in the 3′ non-coding region.
1. A method of establishing whether a biological sample contains WNV (West Nile virus), said method comprising the steps of:
(a) obtaining nucleic acids from the biological sample;
(b) performing an in vitro nucleic acid amplification reaction that comprises nucleic acids obtained in step (a) as templates,
whereby a nucleic acid strand is synthesized if nucleic acids obtained in step (a) comprise WNV nucleic acids,
wherein one end of said nucleic acid strand terminates with the nucleotide sequence SEQ ID NO:74 or the complement thereof, allowing for substitution of RNA and DNA equivalent bases,
wherein said in vitro nucleic acid amplification reaction comprises an oligonucleotide that is up to 60 nucleotides in length and comprises the base sequence of SEQ ID NO:116 or the complement thereof, and
wherein a nucleic acid duplex comprising said nucleic acid strand and said oligonucleotide is formed by complementary base pairing in said in vitro nucleic acid amplification reaction if said nucleic acid strand was synthesized;
(c) determining whether said nucleic acid duplex formed in said in vitro nucleic acid amplification reaction; and
(d) establishing either that,
(i) the biological sample contains WNV if said nucleic acid duplex is determined to have formed in said in vitro nucleic acid amplification reaction, or
(ii) the biological sample does not contain detectable amounts of WNV if said nucleic acid duplex is not determined to have formed in said in vitro nucleic acid amplification reaction.
2. The method of claim 1 , wherein the complement of SEQ ID NO:74 is a template for enzymatic synthesis of nucleic acid in said in vitro nucleic acid amplification reaction of step (b), and wherein the base sequence of said nucleic acid strand comprises the base uracil in place of the base thymine.
3. The method of claim 1 , wherein the biological sample comprises a product of donated blood.
4. The method of claim 3 , wherein the product of donated blood is selected from the group consisting of: a plasma sample, and a serum sample.
5. The method of claim 3 , wherein step (a) comprises capturing nucleic acids onto magnetically attractable particles.
6. The method of claim 5 , wherein said nucleic acid strand synthesized in step (b) comprises the base uracil in place of the base thymine.
7. The method of claim 3 , wherein the in vitro nucleic acid amplification reaction in step (b) comprises one enzyme that provides both reverse transcriptase and DNA polymerase activities, and does not comprise independent DNA polymerase and reverse transcriptase enzymes.
8. The method of claim 7 , wherein the complement of SEQ ID NO:74 is a template for enzymatic synthesis of nucleic acid in said in vitro nucleic acid amplification reaction of step (b), and wherein the base sequence of said nucleic acid strand comprises the base uracil in place of the base thymine.
9. The method of claim 7 , wherein step (c) comprises determining, while the in vitro nucleic acid amplification reaction is occurring, whether said nucleic acid duplex formed.
10. The method of claim 1 , wherein step (a) comprises capturing nucleic acids onto magnetically attractable particles.
11. The method of claim 10 , wherein the in vitro nucleic acid amplification reaction in step (b) comprises one enzyme that provides both reverse transcriptase and DNA polymerase activities, and does not comprise independent DNA polymerase and reverse transcriptase enzymes.
12. The method of claim 11 , wherein step (c) comprises determining, while the in vitro nucleic acid amplification reaction is occurring, whether said nucleic acid duplex formed.
13. The method of claim 1 , wherein the in vitro nucleic acid amplification reaction in step (b) comprises one enzyme that provides both reverse transcriptase and DNA polymerase activities, and does not comprise independent DNA polymerase and reverse transcriptase enzymes.
14. The method of claim 1 , wherein step (c) comprises performing either fluorimetry or luminometry to quantify any of an optical signal that indicates formation of said nucleic acid duplex.
15. The method of claim 1 , wherein step (c) comprises performing fluorimetry, while the in vitro nucleic acid amplification reaction is occurring, to quantify any of an optical signal that indicates formation of said nucleic acid duplex.