Compositions for use in identification of alphaviruses

• US 20100035227A1
• Filed: 03/02/2005
• Published: 02/11/2010
• Est. Priority Date: 03/03/2004
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Sampath, Rangarajan, Hall, Thomas A., Eshoo, Mark W.
• Granted Patent: US 8,119,336 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search
• CPC Class Codes: G16B 40/00 ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
• CPC Class Codes: G16B 40/10 Signal processing, e.g. from mass spectrometry [MS] or from PCR

Abstract

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.

Claims

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 17-28 nucleobases in length comprising at least a subsequence of consecutive nucleobases at least 70% sequence identity with SEQ ID NO:

93 or its complement and a reverse primer 19-35 bases in length or its complement, said conserved regions flanking a variable region that varies between said two or more members of said alphavirus genus wherein upon amplification of a nucleic acid from said alphavirus genus said primer pair generates a bioagent amplicon between 45 consecutive nucleobases in length and 200 consecutive nucleobases in length.

4. The composition of claim 1 wherein said reverse primer comprises at least 70% sequence identity with SEQ ID NO:

66.

5. The composition of claim 1 wherein either or both of said oligonucleotide primers comprises at least one modified nucleobase.

6. The composition of claim 1 wherein either or both of said oligonucleotide primers comprises a non-templated T residue on the 5′

end.

7. The composition of claim 1 wherein either or both of said oligonucleotide primers comprises at least one non-template tag.

8. The composition of claim 1 wherein either or both of said oligonucleotide primers comprises at least one molecular mass modifying tag.

9. A kit comprising the composition of claim 1.

10. The kit of claim 9 further comprising at least one calibration polynucleotide.

11. The kit of claim 9 further comprising at least one ion exchange resin linked to magnetic beads.

18. The composition of claim 1, wherein said forward primer is SEQ ID NO:

21.

2. (canceled)

3. (canceled)

12-17. -17. (canceled)