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

Abstract

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.

Keywords: Monkeypox virus; Real-time PCR; Varicella-zoster virus; Variola virus.

Fig. 1

Sequence alignments of target regions for the VARV ORF B12R (A), MPXV ORF F3L (B) and VZV ORF38 (C). The alignments of 169 (A), 49 (B) and 178 (C) strains were made using BioEdit Sequence Alignment Editor. Strains within one species with identical target region were condensed (the number in parentheses represents the number of strains included). Dotes represent the identical nucleotides in the compared sequences of virus genomes relative to the reference sequences (A: VARV strain India-1967, B: MPXV strain Zaire-96-I-16 and C: VZV strain Ellen); dashes represent the nucleotide deletions. The nucleotide positions in analyzed DNA segments are shown above the nucleotide sequences (positions according to the virus DNA: A, 161552–161671 bp according to the VARV India-1967 strain; B, 48362–48440 bp according to the MPXV Zaire-96-I-16 strain; C, 69295–69383 bp according to the VZV Ellen strain). A: UPPER, VARV_B12R_upper; PROBE, VARV_B12R_probe; LOWER, VARV_B12R_lower. B: UPPER, MPXV_F3L_upper; PROBE, MPXV_F3L_probe; LOWER, MPXV_F3L_lower. C: UPPER, VZV_ORF38_upper; PROBE, VZV_ORF38_probe; LOWER, VZV_ORF38_lower.

Fig. 2

The dependence of fluorescence signal on the number of cycles in real-time PCR. The data were obtained using a Real-Time PCR System 7500 (Applied Biosystems) device with oligonucleotide primers and hybridization probes calculated for species-specific identification of VARV, MPXV, VZV using recombinant plasmids pVARV-B12R, pMPXV-F3L and pVZV-ORF38 (Table 2) (tenfold quantities of copies per reaction are marked). Data are shown for each optical channel used for fluorescence detection: the signal of FAM dye conjugated with VARV-specific hybridization probe, 518 nm; the signal of JOE conjugated with MPXV-specific hybridization probe, 548 nm; the signal of TAMRA conjugated with VZV-specific hybridization probe, 580 nm. Cycle Number – the number of cycles in real-time PCR. Fluorescence – the value of fluorescence signal.

Fig. 3

The dependence of fluorescence signal on the number of cycles in 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 VARV, MPXV, VZV. DNA samples were extracted from experimental specimens (spleen (Ct 22.1), lungs (Ct 19.8), liver (Ct 22.5), blood (Ct 25.9), oral swabs (Ct 20.5), pustules (Ct 19.2)) from marmots infected with MPXV strain CDC#V79-I-005. The data for 548 nm optical channel used for fluorescence detection of JOE conjugated with MPXV-specific hybridization probe are presented. Cycle Number – the number of cycles in real-time PCR. Fluorescence – the value of fluorescence signal.