Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents

Journal of Virological Methods
M AitichouSofi Ibrahim

Abstract

A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6-carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where...Continue Reading

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Citations

Jun 4, 2011·Journal of Virological Methods·Sergei N ShchelkunovElena V Gavrilova
Apr 22, 2009·Journal of Medical Virology·Itoe IizukaShigeru Morikawa
Jan 26, 2017·Scientific Reports·Alexander NagyMartina Havlíčková
Sep 21, 2018·Frontiers in Public Health·Nikola Sklenovská, Marc Van Ranst
Jan 30, 2020·Viruses·Hermann MeyerGeoffrey L Smith

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