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 - 4 . (canceled) 
     
     
         5 . A method for identification of an unknown orthopoxvirus comprising:
 amplifying nucleic acid from said orthopoxvirus using the composition of claim  4  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.   
     
     
         6 . 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 claim  4  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.   
     
     
         7 . A method for determination of the quantity of an unknown orthopoxvirus in a sample comprising:
 contacting said sample with the composition of claim  4  and a known quantity of a calibration polynucleotide comprising a calibration sequence;   concurrently amplifying nucleic acid from said orthopoxvirus in said sample with the composition of claim  4  and amplifying nucleic acid from said calibration polynucleotide in said sample with the composition of claim  4  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.   
     
     
         8 . The method of  claim 7  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: 1:24, 2:25, 3:26, 5:28, 6:29, or 7:30. 
     
     
         9 . The method of  claim 7  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: 1:24, 2:25, 3:26, 5:28, 6:29, or 7:30. 
     
     
         10 . The method of  claim 7  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: 1:24, 2:25, 3:26, 5:28, 6:29, or 7:30. 
     
     
         11 . The method of  claim 7  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: 1:24, 2:25, 3:26, 5:28, 6:29, or 7:30. 
     
     
         12 - 18 . (canceled)