Compositions for use in identification of alphaviruses

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.

1 .- 11 . (canceled) 
     
     
         12 . A method for identification of an unknown alphavirus comprising:
 amplifying nucleic acid from said alphavirus using 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 to obtain an amplification product;   measuring the molecular mass of said amplification product;   optionally, determining the base composition of said amplification product from said molecular mass; and   comparing said molecular mass or base composition with a plurality of molecular masses or base compositions of known alphaviral bioagent identifying amplicons, wherein a match between said molecular mass or base composition and a member of said plurality of molecular masses or base compositions identifies said unknown alphavirus.   
     
     
         13 . The method of  claim 12  wherein said molecular mass is measured by mass spectrometry. 
     
     
         14 .- 15 . (canceled) 
     
     
         16 . A method for determination of the quantity of an unknown alphavirus in a sample comprising:
 contacting said sample with 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 and a known quantity of a calibration polynucleotide comprising a calibration sequence;   concurrently amplifying nucleic acid from said alphavirus in said sample with said composition and amplifying nucleic acid from said calibration polynucleotide in said sample with said composition to obtain a first amplification product comprising an alphaviral bioagent identifying amplicon and a second amplification product comprising a calibration amplicon;   determining the molecular mass and abundance for said alphaviral bioagent identifying amplicon and said calibration amplicon; and   distinguishing said alphaviral bioagent identifying amplicon from said calibration amplicon based on molecular mass, wherein comparison of alphaviral bioagent identifying amplicon abundance and calibration amplicon abundance indicates the quantity of alphavirus in said sample.   
     
     
         17 . The method of  claim 16  further comprising determining the base composition of said alphaviral bioagent identifying amplicon. 
     
     
         18 . The composition of  claim 12  wherein either or both of said oligonucleotide primers comprises at least one modified nucleobase. 
     
     
         19 . The composition of  claim 12  wherein either or both of said oligonucleotide primers comprises a non-templated T residue on the 5′ end. 
     
     
         20 . The composition of  claim 12  wherein either or both of said oligonucleotide primers comprises at least one non-template tag. 
     
     
         21 . The composition of claim  1  wherein either or both of said oligonucleotide primers comprises at least one molecular mass modifying tag. 
     
     
         22 . A method for identification of an unknown alphavirus comprising:
 amplifying nucleic acid from said alphavirus using 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 to obtain an amplification product;   measuring the molecular mass of said amplification product;   optionally, determining the base composition of said amplification product from said molecular mass; and   comparing said molecular mass or base composition with a plurality of molecular masses or base compositions of known alphaviral bioagent identifying amplicons, wherein a match between said molecular mass or base composition and a member of said plurality of molecular masses or base compositions identifies said unknown alphavirus.