Species-specific identification of variola, monkeypox, cowpox, and vaccinia viruses by multiplex real-time PCR assay

A method of one-stage rapid identification of variola (VARV), monkeypox (MPXV), cowpox (CPXV), and vaccinia (VACV) viruses, pathogenic for humans, utilizing multiplex real-time TaqMan PCR (MuRT-PCR) assay was developed. Four pairs of oligonucleotide primers and four hybridization probes with various fluorescent dyes and the corresponding fluorescence quenchers were concurrently used for MuRT-PCR assay. The hybridization probe specific for the VARV sequence contained FAM/BHQ1 as a dye/quencher pair; MPXV-specific, TAMRA/BHQ2; CPXV-specific, JOE/BHQ1; VACV-specific, Cy5/BHQ3. The specificity and sensitivity of the developed method were assessed by analyzing DNA of 29 strains belonging to six orthopoxvirus species as well as the DNA samples isolated from archive clinical specimens of human smallpox cases and experimental specimens isolated from CPXV-infected mice and MPXV-infected marmot.