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

Abstract

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.

Fig. 1

Comparison of the ORF A38R nucleotide sequence of VARV strains India-1967 (VARV-IND), Garcia-1966 (VARV-GAR), and Bangladesh-1975 (VARV-BSH) with the corresponding regions of MPXV strains Zaire-96-I-16 (MPXV-ZAI) and USA_2003_039 (MPXV-USA); CPXV strains GRI-90 (CPXV-GRI) and Brighton Red (CPXV-BRI); VACV strains Copenhagen (VACV-COP), and Ankara (VACV-ANK); CMLV strains CMS (CMLV-CMS) and M-96 (CMLV-M96); ECTV strains Naval (ECTV-NAV) and Moscow (ECTV-MOS). The identical nucleotides in the compared sequences of virus genomes relative to the VARV-IND sequence are denoted with dots and the deletions, with dashes. The nucleotide positions in analyzed DNA segment are shown to the left and right of nucleotide sequence. The sequences of oligonucleotide primers VARV_A38R_forward and VARV_A38R_reverse and hybridization probe VARV_A38R_probe for fluorescence detection of VARV DNA are shown in boldfaced italic.

Fig. 2

The dependences of fluorescence signal on the number of cycles in multiplex real-time PCR. The data were obtained using a Real-Time PCR System 7500 (Applied Biosystems) device with the oligonucleotide primers and hybridization probes calculated for species-specific identification of orthopoxviruses (Table 2) and are shown for each optical channel used for fluorescence detection: (A) the signal of FAM dye conjugated with VARV-specific hybridization probe, 518 nm; (B) the signal of TAMRA conjugated with MPXV-specific hybridization probe, 580 nm; (C) the signal of Cy5 conjugated with VACV-specific hybridization probe, 643 nm; (D) the signal of JOE conjugated with CPXV-specific hybridization probe, 548 nm. The amplicons produced from DNAs of 29 strains belonging to six orthopoxvirus species and several control DNAs (Table 1) were analyzed simultaneously for 4 optical channels noted above. The lines above the horizontal band correspond to positive results and reflect accumulation level of the PCR products characteristic of each analyzed orthopoxvirus species (A–D) and the lines below the horizontal band, to the negative results for the remaining poxvirus and herpesvirus species. Cycle Number, the number of cycles in real-time PCR and Rn, the value of fluorescence signal.