title: Multicolour, multiplex real-time PCR assay for the detection of human-pathogenic poxviruses.
abstract: After eradication of variola virus, one of the most dangerous infectious diseases affecting mankind, today other poxviruses of different genera can cause infection in humans. These viruses include human-specific molluscipoxviruses as well as zoonotic orthopoxviruses and parapoxviruses. While non-variola orthopoxvirus infections mostly cause mild symptoms in immunocompetent persons, they can evoke severe disease in immunocompromised patients. Since the typical poxviral skin lesions are rarely diagnosed by physicians, PCR-based identification of suspected poxviruses is often required. To simplify the PCR-based diagnosis of human-pathogenic poxviruses, we established a multicolour multiplex real-time PCR that simultaneously detects and differentiates human-pathogenic poxviruses in one reaction. Using 5' nuclease probes labelled with FAM for orthopoxviruses, VIC for parapoxviruses and FAM and VIC for molluscipoxviruses, respectively, amplification of poxviral DNA resulted in a genus-specific reporter-dye profile. Validation with 36 human clinical specimens and DNA of pathogens causing pox-like skin lesions demonstrated the specificity of the assay. Probit analysis revealed a limit of detection of 9.7, 22.08 and 28.1 copies/assay (95% CI) for molluscipoxvirus, orthopoxvirus and parapoxvirus DNA, respectively. The combinatorial multicolour strategy applied has the potential to be used in further applications targeting even more than three pathogens.
authors.affiliation: Robert Koch-Institut, Centre for Biological Safety 1, Nordufer 20, 13353 Berlin, Germany.
authors.forename: Kati
authors.initials: K
authors.lastname: Schroeder
authors.forename: Andreas
authors.initials: A
authors.lastname: Nitsche
mesh_terms: D001681:Biological Assay / Q000379:methods*; D006801:Humans; D011212:Poxviridae / Q000302:isolation & purification* / Q000472:pathogenicity*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*