Compositions for use in identification of alphaviruses
View Patent ↗The present invention provides oligonucleotide primers and compositions and kits containing the same for rapid identification of alphaviruses by amplification of a segment of viral nucleic acid followed by molecular mass analysis.
1. A composition comprising a purified oligonucleotide primer pair configured to generate amplicons from two or more members of the alphavirus genus by hybridizing a forward primer and a reverse primer to conserved regions of a nsP1 encoding gene in two or more members of said alphavirus genus, said primer pair comprising a forward primer with 100% sequence identity with SEQ ID NO: 93 and a reverse primer with 100% sequence identity with SEQ ID NO: 66.
2. The composition of claim 1 wherein either or both of said oligonucleotide primers comprises at least one modified nucleobase.
3. The composition of claim 1 wherein either or both of said oligonucleotide primers comprises a non-templated T residue on the 5′ end.
4. The composition of claim 1 wherein either or both of said oligonucleotide primers comprises at least one non-template tag.
5. The composition of claim 1 wherein either or both of said oligonucleotide primers comprises at least one molecular mass modifying tag.
6. A kit comprising the composition of claim 1 .
7. The kit of claim 6 further comprising at least one calibration polynucleotide.
8. The kit of claim 6 further comprising at least one ion exchange resin linked to magnetic beads.
9. A composition comprising a purified oligonucleotide primer pair configured to generate amplicons from two or more members of the alphavirus genus by hybridizing a forward primer and a reverse primer to conserved regions of a nsP1 encoding gene in two or more members of said alphavirus genus, said primer pair comprising a forward primer with 100% sequence identity with SEQ ID NO: 21 and a reverse primer with 100% sequence identity with SEQ ID NO: 66.
10. A method for generating alphavirus amplicons comprising:
1) providing a composition according to claim 1 ; and
2) contacting an alphavirus nucleic acid with the composition to generate alphavirus amplicons.