Westcott David G, King Donald P, Drew Trevor W, Nowotny Norbert, Kindermann Johanna, Hannant Duncan, Belák Sándor, Paton David J
Department of Virology, Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey, KT15 3NB, UK.
Vet Res. 2003 Mar-Apr;34(2):165-76. doi: 10.1051/vetres:2002063.
Routine detection of equine arteritis virus (EAV) can be achieved by virus isolation (VI) in cell culture, or by the amplification of viral genome by molecular methods. To simplify molecular diagnosis, a number of different Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and RT-nested PCR (RT-nPCR) assays were compared, and a one-tube method was developed and optimised utilizing a fluorogenic probe (TaqMan). An artificial RNA template (Mimic) and associated probe were also constructed to provide in-tube validation of the RT-PCR system. To assess the utility of the RT-PCR TaqMan assay, 28 different isolates of EAV representing different genetic groups of American and European strains were tested. Furthermore, the ability of VI and RT-PCR TaqMan assay to detect EAV in different biological matrices such as semen, nasal and faecal swabs and blood was compared. All 28 EAV strains were detected by the RT-PCR TaqMan assay. The results of TaqMan and VI testing were in agreement for 30 of the 33 semen samples and all of the 50 other clinical specimens examined: the RT-PCR TaqMan assay detected 18 positive semen samples, three more than VI. In conclusion, the one-tube RT-PCR TaqMan assay is a rapid, reliable method for the detection of EAV.
马动脉炎病毒（EAV）的常规检测可通过在细胞培养中进行病毒分离（VI），或通过分子方法扩增病毒基因组来实现。为简化分子诊断，比较了多种不同的逆转录聚合酶链反应（RT-PCR）和RT巢式PCR（RT-nPCR）检测方法，并利用荧光探针（TaqMan）开发并优化了一种单管方法。还构建了人工RNA模板（模拟物）和相关探针，以对RT-PCR系统进行管内验证。为评估RT-PCR TaqMan检测方法的实用性，对代表美国和欧洲不同遗传组的28种不同的EAV分离株进行了检测。此外，还比较了VI和RT-PCR TaqMan检测方法在精液、鼻拭子、粪便拭子和血液等不同生物基质中检测EAV的能力。RT-PCR TaqMan检测方法检测出了所有28种EAV毒株。在检测的33份精液样本中的30份以及所有50份其他临床标本中，TaqMan检测结果与VI检测结果一致：RT-PCR TaqMan检测方法检测出18份精液阳性样本，比VI检测多3份。总之，单管RT-PCR TaqMan检测方法是一种快速、可靠的EAV检测方法。
