title: Real-time PCR assay for specific detection of cowpox virus.
abstract: The species cowpox virus (CPXV), genus Orthopoxvirus (OPV), consists of isolates highly variable in their biological properties and their genotypes. A TaqMan PCR assay for the specific detection of CPXV DNA based on sequences of the ORF D11L has been developed recently. (Gavrilova et al., 2010; Shchelkunov et al., 2011); however, a rather limited panel of CPXV stains has been used. When a much larger panel of 47 CPXV DNAs has been tested, three strains could not be amplified at all because of large deletions in their respective ORF D11L. In addition, a deletion of 23bp led to low-efficiency detection of five other CPXV strains. To solve this problem a new primer/probe combinations was selected based on sequences of ORF D8L, and a new real-time PCR method for (i) a genus-specific detection of OPVs and (ii) a simultaneous CPXV-specific differentiation is described in this study. The specificity and sensitivity were assessed by analyzing DNA of 67 strains belonging to human-pathogenic OPV species, including variola virus, as well as specimens of CPXV-infected mice.
authors.affiliation: State Research Center of Virology and Biotechnology "VECTOR", Koltsovo, Novosibirsk Region 630559, Russia. Electronic address: maksrinat@yandex.ru.
authors.forename: Rinat A
authors.initials: RA
authors.lastname: Maksyutov
authors.affiliation: State Research Center of Virology and Biotechnology "VECTOR", Koltsovo, Novosibirsk Region 630559, Russia. Electronic address: lgavrilova@yandex.ru.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Gavrilova
authors.affiliation: Institut für Mikrobiologie der Bundeswehr, München 80937, Germany. Electronic address: Hermann1Meyer@Bundeswehr.org.
authors.forename: Hermann
authors.initials: H
authors.lastname: Meyer
authors.affiliation: State Research Center of Virology and Biotechnology "VECTOR", Koltsovo, Novosibirsk Region 630559, Russia. Electronic address: snshchel@vector.nsc.ru.
authors.forename: Sergei N
authors.initials: SN
authors.lastname: Shchelkunov
mesh_terms: D000818:Animals; D015608:Cowpox virus / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D014644:Genetic Variation; D051379:Mice; D015345:Oligonucleotide Probes / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*
chemical_list: D017931:DNA Primers; D015345:Oligonucleotide Probes
keywords: Cowpox virus; Detection; Orthopoxvirus; Real-time PCR