Marston Denise A, Jennings Daisy L, MacLaren Nikki C, Dorey-Robinson Daniel, Fooks Anthony R, Banyard Ashley C, McElhinney Lorraine M
Wildlife Zoonoses & Vector-Borne Diseases Research Group, Animal and Plant Health Agency;
Wildlife Zoonoses & Vector-Borne Diseases Research Group, Animal and Plant Health Agency.
J Vis Exp. 2019 Jul 10(149). doi: 10.3791/59709.
Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to confirm rabies diagnosis but have only recently been accepted by the OIE (World Organisation for Animal Health) as a primary method to detect rabies infection. Real-time RT-PCR assays provide real-time data, and are closed-tube systems, minimizing the risk of contamination during setup. DNA intercalating fluorochrome real-time RT-PCR assays do not require expensive probes, minimizing the cost per sample, and when the primers are designed in conserved regions, assays that are specific across virus genera rather than specific to just one virus species are possible. Here we describe a pan-lyssavirus SYBR real-time RT-PCR assay that detects lyssaviruses across the Lyssavirus genus, including the most divergent viruses IKOV, WCBV and LLEBV. In conjunction with dissociation curve analysis, this assay is sensitive and specific, with the advantage of detecting all lyssavirus species. The assay has been adopted in many diagnostic laboratories with quality assured environments, enabling robust, rapid, sensitive diagnosis of animal and human rabies cases.
分子检测方法快速、灵敏且特异，已成为狂犬病诊断的核心手段。基于聚合酶链反应（PCR）的检测方法已应用数十年以确诊狂犬病，但直到最近才被国际兽疫局（世界动物卫生组织）认可为检测狂犬病感染的主要方法。实时逆转录PCR（RT-PCR）检测方法可提供实时数据，并且是闭管系统，可将检测设置过程中的污染风险降至最低。DNA嵌入荧光染料实时RT-PCR检测方法无需昂贵的探针，从而将每个样本的成本降至最低，而且当引物设计在保守区域时，就有可能实现针对整个病毒属而非仅针对一种病毒物种的特异性检测。在此，我们描述了一种泛狂犬病病毒属SYBR实时RT-PCR检测方法，该方法可检测狂犬病病毒属中的所有狂犬病病毒，包括差异最大的病毒IKOV、WCBV和LLEBV。结合熔解曲线分析，该检测方法灵敏且特异，具有检测所有狂犬病病毒物种的优势。该检测方法已在许多具备质量保证环境的诊断实验室中采用，能够对动物和人类狂犬病病例进行可靠、快速、灵敏的诊断。
