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

Autor: Kati Schroeder, Andreas Nitsche
Rok vydání: 2010
Předmět:
Zdroj: Molecular and Cellular Probes. 24:110-113
ISSN: 0890-8508
DOI: 10.1016/j.mcp.2009.10.008
Popis: 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.
Databáze: OpenAIRE
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