Amplification refractory mutation system PCR assays for the detection of variola and Orthopoxvirus.
PCR assays that can identify the presence of variola virus (VARV) sequences in an unknown DNA sample were developed using principles established for the amplification refractory mutation system (ARMS). The assay's specificity utilised unique single nucleotide polymorphisms (SNP) identified among Orthopoxvirus (OPV) orthologs of the vaccinia virus Copenhagen strain A13L and A36R genes. When a variola virus specific primer was used with a consensus primer in an ARMS assay with different Orthopoxvirus genomes, a PCR product was only amplified from variola virus DNA. Incorporating a second consensus primer into the assay produced a multiplex PCR that provided Orthopoxvirus generic and variola-specific products with variola virus DNA. We tested two single nucleotide polymorphisms with a panel of 43 variola virus strains, collected over 40 years from countries across the world, and have shown that they provide reliable markers for variola virus identification. The variola virus specific primers did not produce amplicons with either assay format when tested with 50 other Orthopoxvirus DNA samples. Our analysis shows that these two polymorphisms were conserved in variola virus genomes and provide a reliable signature of Orthopoxvirus species identification.
Pulford
David
D
Biomedical Sciences, DSTL Porton Down, Salisbury, Wiltshire SP4 0JQ, UK. davidpulford@hotmail.com
Meyer
Hermann
H
Brightwell
Gale
G
Damon
Inger
I
Kline
Richard
R
Ulaeto
David
D
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005784:Gene Amplification; D005843:Geography; D006801:Humans; D008969:Molecular Sequence Data; D018155:Orthopoxvirus / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012899:Smallpox / Q000175:diagnosis* / Q000662:veterinary; D012901:Variola virus / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; D004279:DNA, Viral