title: Development of real-time PCR assay for specific detection of cowpox virus.
abstract: BACKGROUND
  The number of recorded human cowpox cases are recently increasing. The symptoms caused by cowpox virus (CPXV) in a number of human cases are close to the symptoms characteristic of the orthopoxviral human infections caused by monkeypox or smallpox (variola) viruses. Any rapid and reliable real-time PCR method for distinguishing cowpox from smallpox and monkeypox is yet absent.
  
  
  OBJECTIVE
  The aim of this study was to develop a quick and reliable real-time TaqMan PCR assay for specific detection of cowpox virus and to determine the sensitivity and specificity of this method.
  
  
  METHODS
  Based on aligned nucleotide sequences of orthopoxviruses, we found a virus-specific region in the CPXV genome and selected the oligonucleotide primers and hybridization probe within this region. The specificity of the developed method was tested using a panel of various orthopoxvirus (OPV) DNAs. The sensitivity was determined using the recombinant plasmid carrying a fragment of CPXV DNA and genomic DNA of the CPXV strain GRI-90.
  
  
  RESULTS
  The analytical specificity of this method was determined using DNAs of 17 strains of four OPV species pathogenic for humans and amounted to 100%. The method allows 6 copies of plasmid DNA and 20 copies of CPXV DNA in the reaction mixture to be detected.
  
  
  CONCLUSIONS
  A quick and reliable TaqMan PCR assay providing for a highly sensitive and specific detection of CPXV DNA was developed.
authors.affiliation: SRC VB "Vector", Koltsovo, Novosibirsk Region 630559, Russia. lgavrilova@yandex.ru
authors.forename: Elena V
authors.initials: EV
authors.lastname: Gavrilova
authors.forename: Dmitrii N
authors.initials: DN
authors.lastname: Shcherbakov
authors.forename: Rinat A
authors.initials: RA
authors.lastname: Maksyutov
authors.forename: Sergei N
authors.initials: SN
authors.lastname: Shchelkunov
mesh_terms: D000818:Animals; D015605:Cowpox / Q000175:diagnosis / Q000821:virology; D015608:Cowpox virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D006801:Humans; D018155:Orthopoxvirus / Q000145:classification / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011213:Poxviridae Infections / Q000175:diagnosis / Q000821:virology*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D004279:DNA, Viral