Development of real-time PCR assay for specific detection of cowpox virus.
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.
Gavrilova
Elena V
EV
SRC VB "Vector", Koltsovo, Novosibirsk Region 630559, Russia. lgavrilova@yandex.ru
Shcherbakov
Dmitrii N
DN
Maksyutov
Rinat A
RA
Shchelkunov
Sergei N
SN
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
D017931:DNA Primers; D004279:DNA, Viral