Species-specific differentiation of variola, monkeypox, and varicella-zoster viruses by multiplex real-time PCR assay

•Similarity of the clinical manifestations of monkeypox with chickenpox.•Multiplex real-time TaqMan PCR assay was developed.•Variola, monkeypox and varicella-zoster virus differentiation. A method of one-stage rapid detection and differentiation of epidemiologically important variola virus (VARV), monkeypox virus (MPXV), and varicella-zoster virus (VZV) utilizing multiplex real-time TaqMan PCR assay was developed. Four hybridization probes with various fluorescent dyes and the corresponding fluorescence quenchers were simultaneously used for the assay. The hybridization probes specific for the VARV sequence contained FAM/BHQ1 as a dye/quencher pair; MPXV-specific, JOE/BHQ1; VZV-specific, TAMRA/BHQ2; and internal control-specific, Cy5/BHQ3. The specificity and sensitivity of the developed method were assessed by analyzing DNA of 32 strains belonging to orthopoxvirus and herpesvirus species.