Dengue virus assay
View Patent ↗Nucleic acid assays for detecting nucleic acids of Dengue virus serotypes 1-4 derived from 5′ NTR.
1. A method of determining whether a test sample contains Dengue virus, said method comprising the steps of:
(a) obtaining nucleic acids from the test sample;
(b) performing an in vitro nucleic acid amplification reaction using the nucleic acids obtained in step (a) as templates for amplification with a set of primers, whereby there is produced an amplification product if said test sample comprised nucleic acids of any of Dengue virus serotypes 1-4,
wherein the base sequence of a first member of said set of primers consists of a target-hybridizing sequence of SEQ ID NO:2, optionally joined to an upstream promoter sequence, and further optionally joined to an upstream tag sequence, wherein neither said upstream promoter sequence nor said upstream tag sequence can hybridize to the nucleic acid of any of Dengue virus serotypes 1-4 and participate in said in vitro nucleic acid amplification reaction in the absence of joining to said target-hybridizing sequence, and
wherein the base sequence of a second member of said set of primers consists of SEQ ID NO:14; and
(c) detecting, with a first detectably labeled hybridization probe, any of said amplification product that may have been produced in the in vitro nucleic acid amplification reaction, wherein the base sequence of said first detectably labeled hybridization probe is SEQ ID NO:22 or the complement thereof, allowing for substitution of RNA and DNA equivalent bases,
whereby detecting said amplification product determines that the test sample contains at least one of Dengue virus serotypes 1-4, and
whereby failing to detect said amplification product determines that the test sample does not contain Dengue virus.
2. The method of claim 1 , wherein obtaining step (a) comprises capturing nucleic acids from the test sample onto a solid support, and then isolating the solid support.
3. The method of claim 1 , wherein the in vitro nucleic acid amplification reaction in performing step (b) is an isothermal in vitro nucleic acid amplification reaction.
4. The method of claim 1 , wherein detecting step (c) comprises detecting by a procedure selected from the group consisting of luminometry and fluorometry.
5. The method of claim 1 , wherein detecting step (c) comprises detecting a chemiluminescent signal by luminometry.
6. The method of claim 1 , wherein the base sequence of said first member of said set of primers in performing step (b) consists of said target-hybridizing sequence of SEQ ID NO:2 joined to said upstream promoter sequence.
7. The method of claim 1 , wherein detecting step (c) comprises measuring an optical signal produced by said detectably labeled hybridization probe, and then comparing the measured optical signal to a cutoff value.
8. The method of claim 1 , wherein the test sample is selected from the group consisting of blood, blood products, and serum.
9. The method of claim 6 , wherein said set of primers further comprises a third member, and wherein the base sequence of said third member of said set of primers is SEQ ID NO:15.
10. The method of claim 1 , wherein said set of primers further comprises a third member, and wherein the base sequence of said third member of said set of primers is SEQ ID NO:15.
11. The method of claim 6 , further comprising a second detectably labeled hybridization probe, wherein the base sequence of said second detectably labeled hybridization probe is SEQ ID NO:23 or the complement thereof, allowing for substitution of RNA and DNA equivalent bases.
12. The method of claim 1 , further comprising a second detectably labeled hybridization probe, wherein the base sequence of said second detectably labeled hybridization probe is SEQ ID NO:23 or the complement thereof, allowing for substitution of RNA and DNA equivalent bases.