Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology · 2010
Development of real-time PCR assay for specific detection of cowpox virus.
Оценочное исследованиеСтатьяНазвание и аннотация: Перевод в очереди
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Gavrilova Elena V, Shcherbakov Dmitrii N, Maksyutov Rinat A, Shchelkunov Sergei N
Аннотация
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.
OBJECTIVES: 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.
STUDY DESIGN: 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.
CONCLUSION: A quick and reliable TaqMan PCR assay providing for a highly sensitive and specific detection of CPXV DNA was developed.
OBJECTIVES: 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.
STUDY DESIGN: 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.
CONCLUSION: A quick and reliable TaqMan PCR assay providing for a highly sensitive and specific detection of CPXV DNA was developed.