猴痘、天花病毒感染快速分子诊断荧光定量实时PCR方法的建立

Rapid Detection and Differentiation of Smallpox or Monkeypox Virus Infection by Real-Time PCR Assay

目的:天花、猴痘可感染人并引起严重皮疹、发热等临床症状,均为烈性传染病,是潜在的生物恐怖因子,因此需要建立针对其感染的快速特异的诊断方法。方法:分别设计正痘病毒属通用型、天花病毒特异、猴痘病毒特异的引物与荧光标记探针,建立荧光定量实时PCR方法,对人工合成或模拟样本进行检测。结果:可在4h内对天花或猴痘病毒感染进行特异性鉴别诊断,检测灵敏度可达100拷贝/25μL反应体积。结论:本方法可作为一种检疫与反恐应急储备技术。

Objective： The orthopox viruses that are high pathogenic for human include variola virus（VARV, smallpox virus） and monkeypox virus（MPV）. Either is considered as a potential threat agent or bioterrorist weapon. The special and sensitive detector is needed. Methods： The pan-orthopox, VARV-specific and MPV-specific PCR primers and fluore-cence-labeled probes were designed to develop a real-time PCR assay for rapid identification of smallpox or monkeypox virus infection. Results： The results showed that these real-time assays could detect and differentiate smallpox or monkey-pox virus infection rapidly （less than 4 hours） and the detection limits were 10 copies of target DNA per PCR vial when the target genes were inserted in the plasmid or vaccinia virus（Tiantan strain） as the PCR templates. Conclusion： This assay may represent a battery of tests in situations of both natural import and potential acts of bioterrorism.