Multiplex PCR detection and species differentiation of orthopoxviruses pathogenic to humans

Abstract

A method for one-stage rapid identification of four orthopoxvirus species pathogenic to humans based on multiplex polymerase chain reaction (MPCR) was developed. Five pairs of oligonucleotide primers--one, genus-specific; and the rest, species-specific for variola, monkeypox, cowpox, and vaccinia viruses, respectively--were used concurrently for MPCR assay of orthopoxvirus DNAs. Specificity and sensitivity of the method developed were evaluated using DNAs of 57 orthopoxvirus strains, including the DNAs isolated from human case clinical materials.

Fig. 1

Graphical alignment of (a) left and (b) right species-specific genomic regions of cowpox virus strain GRI-90 (CPXV-GRI), vaccinia virus strain Copenhagen (VACV-COP), monkeypox virus strain ZAI-96 (MPXV-ZAI), and variola virus strain India-1967 (VARV-IND). Arrows indicate directions and lengths of the ORFs wherein species-specific amplification was performed. ORF names are given above the arrows. Black blocks represent coding sequences of viral DNAs. Fine lines represent deletions in viral genomes relative to other viruses; ellipses, terminal hairpins of viral DNAs.

Fig. 2

Graphical alignment of DNA sequences of various orthopoxviruses—cowpox virus strain GRI-90 (CPXV-GRI), monkeypox virus strain ZAI-96 (MPXV-ZAI), vaccinia virus strain Copenhagen (VACV-COP), and variola virus strains India-1967 (VARV-IND) and Garcia-1966 (VARV-GAR)—with the corresponding genomic region of CPXV strain 91/1 (CPXV-91/1), containing ‘variola virus-specific’ sequence. Arrows indicate directions and lengths of the ORFs. ORF names are given above the arrows. Fine lines represent deletions in viral genomes relative to other viruses. P1 and P2 are oligonucleotide primers used for specific amplification of VARV DNAs.

Fig. 3

Electrophoretic separation in 2% agarose of the amplicons produced by MPCR using four pairs of oligonucleotide primers for species-specific identification of orthopoxviruses: (1) VACV strain LIVP; (2) VACV strain Patwadanger; (3) VACV strain CVI-78; (4) CPXV strain OPV-91/1; (5) CPXV strain GRI-90; (6) CPXV strain EP-5; (7) CPXV strain EP-2; (8) CPXV strain OPV-98/5; (9) VARV strain Ind-3a; (10) VARV strain Butler; (11) MPXV strain CDC# v79-I-005; (12) MPXV strain CDC# v78-I-3945; (13) Ectromelia virus strain 4908; (14) negative control; and M, DNA marker (lengths in bp are shown to the right).