Multicolour, multiplex real-time PCR assay for the detection of human-pathogenic poxviruses

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.