Compositions for use in identification of orthopoxviruses

Abstract

Oligonucleotide primers and compositions and kits containing the same for rapid identification of orthopoxviruses by amplification of a segment of viral nucleic acid followed by molecular mass analysis are provided.

1 - 18 . (canceled) 
     
     
         19 . A method for identification of an unknown  orthopoxvirus  comprising:
 amplifying nucleic acid from said  orthopoxvirus  using an oligonucleotide primer 13 to 35 nucleobases in length comprising at least 70% sequence identity with SEQ ID NO: 1 and an oligonucleotide primer 15 to 35 nucleobases in length comprising at least 70% sequence identity with SEQ ID NO: 24 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  orthopoxvirus  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  orthopoxvirus.      
     
     
         20 . A method of determining the presence or absence of an  orthopoxvirus  species in a sample comprising:
 amplifying nucleic acid from said sample using the composition of an oligonucleotide primer 13 to 35 nucleobases in length comprising at least 70% sequence identity with SEQ ID NO: 1 and an oligonucleotide primer 15 to 35 nucleobases in length comprising at least 70% sequence identity with SEQ ID NO: 24 to obtain an amplification product;   determining 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 of said amplification product with the known molecular masses or base compositions of one or more known  orthopoxvirus  species bioagent identifying amplicons, wherein a match between said molecular mass or base composition of said amplification product and the molecular mass or base composition of one or more known  orthopoxvirus  species bioagent identifying amplicons indicates the presence of said  orthopoxvirus  species in said sample.   
     
     
         21 . A method for determination of the quantity of an unknown  orthopoxvirus  in a sample comprising:
 contacting said sample with an oligonucleotide primer 13 to 35 nucleobases in length comprising at least 70% sequence identity with SEQ ID NO: 1 and an oligonucleotide primer 15 to 35 nucleobases in length comprising at least 70% sequence identity with SEQ ID NO: 24 and a known quantity of a calibration polynucleotide comprising a calibration sequence;   concurrently amplifying nucleic acid from said  orthopoxvirus  in said sample with an oligonucleotide primer 13 to 35 nucleobases in length comprising at least 70% sequence identity with SEQ ID NO: 1 and an oligonucleotide primer 15 to 35 nucleobases in length comprising at least 70% sequence identity with SEQ ID NO: 24 and amplifying nucleic acid from said calibration polynucleotide in said sample with an oligonucleotide primer 13 to 35 nucleobases in length comprising at least 70% sequence identity with SEQ ID NO: 1 and an oligonucleotide primer 15 to 35 nucleobases in length comprising at least 70% sequence identity with SEQ ID NO: 24 to obtain a first amplification product comprising an  orthopoxvirus  bioagent identifying amplicon and a second amplification product comprising a calibration amplicon;   determining the molecular mass and abundance for said  orthopoxvirus  bioagent identifying amplicon and said calibration amplicon; and   distinguishing said  orthopoxvirus  bioagent identifying amplicon from said calibration amplicon based on molecular mass, wherein comparison of  orthopoxvirus  bioagent identifying amplicon abundance and calibration amplicon abundance indicates the quantity of  orthopoxvirus  in said sample.   
     
     
         22 . The method of  claim 21  further comprising repeating said steps, wherein a different primer pair is used, wherein each member of said different primer pair is of a length of 13 to 35 nucleobases and comprises 70% to 100% sequence identity with the corresponding member of any of the pairs of primers of SEQ ID NOs: 2:25, 3:26, 5:28, 6:29, or 7:30. 
     
     
         23 . The method of  claim 21  further comprising repeating said steps, wherein two different primer pairs are used, wherein each member of said two different primer pairs is of a length of 13 to 35 nucleobases and comprises 70% to 100% sequence identity with the corresponding member of any of the pairs of primers of SEQ ID NOs: 2:25, 3:26, 5:28, 6:29, or 7:30. 
     
     
         24 . The method of  claim 21  further comprising repeating said steps, wherein three different primer pairs are used, wherein each member of said three different primer pairs is of a length of 13 to 35 nucleobases and comprises 70% to 100% sequence identity with the corresponding member of any of the pairs of primers of SEQ ID NOs: 2:25, 3:26, 5:28, 6:29, or 7:30. 
     
     
         25 . The method of  claim 21  further comprising repeating said steps, wherein four different primer pairs are used, wherein each member of said four different primer pairs is of a length of 13 to 35 nucleobases and comprises 70% to 100% sequence identity with the corresponding member of any of the pairs of primers of SEQ ID NOs: 2:25, 3:26, 5:28, 6:29, or 7:30.