Rodriguez-Sanchez Belen, Fernandez-Pinero Jovita, Sailleau Corinne, Zientara Stephan, Belak Sandor, Arias Marisa, Sanchez-Vizcaino Jose Manuel
Animal Health Department, Veterinary School, Universidad Complutense, Av. Puerta de Hierro s/n, 28040 Madrid, Spain.
J Virol Methods. 2008 Jul;151(1):87-94. doi: 10.1016/j.jviromet.2008.03.029. Epub 2008 May 23.
In order to improve, ensure and accelerate the diagnosis of African horse sickness, a highly devastating, transboundary animal disease listed by the World Animal Health Organisation, (OIE) three novel diagnostic PCR assays were developed and tested in this study. The reverse transcription-PCR (RT-PCR) tests were the following: (a) a conventional, gel-based RT-PCR, (b) a real-time PCR with SYBR-Green-named rRT-PCR SYBR-Green-, and (c) a real-time PCR rRT-PCR with TaqMan probe (termed rRT-PCR TaqMan). The same pair of primers-directed against African Horse Sickness Virus (AHSV) segment 5, encoding the non-structural protein NS1, is used in the three tests listed above. The three PCR assays detected similarly the nine AHSV serotypes from cultivated viral suspensions of different origins. The RT-PCR assays provided high sensitivity ranging from 0.1 to 1.2TCID(50)/ml. The specificity was also high, considering that related viruses, such as Bluetongue virus, and other equine viruses, such as West Nile Virus, remained negative for RT-PCR amplification. The detection of AHSV virus can be completed within 2-3h. These results indicate that the novel PCR methods described in this paper provide robust and versatile tools that allow rapid and highly specific, simultaneous detection of all AHSV serotypes.
为了改进、确保并加速对非洲马瘟的诊断，非洲马瘟是世界动物卫生组织（OIE）列出的一种极具毁灭性的跨界动物疾病，本研究开发并测试了三种新型诊断性聚合酶链反应（PCR）检测方法。逆转录PCR（RT-PCR）检测如下：（a）一种传统的基于凝胶的RT-PCR，（b）一种使用SYBR Green的实时PCR（称为rRT-PCR SYBR Green），以及（c）一种使用TaqMan探针的实时PCR（rRT-PCR TaqMan）。上述三种检测均使用同一对引物，该引物针对编码非结构蛋白NS1的非洲马瘟病毒（AHSV）第5节段。这三种PCR检测方法从不同来源的培养病毒悬液中检测出九种AHSV血清型的效果相似。RT-PCR检测灵敏度高，范围为0.1至1.2TCID(50)/ml。特异性也很高，因为相关病毒如蓝舌病病毒以及其他马病毒如西尼罗河病毒，RT-PCR扩增结果均为阴性。AHSV病毒的检测可在2-3小时内完成。这些结果表明，本文所述的新型PCR方法提供了强大且通用的工具，可实现对所有AHSV血清型的快速、高度特异性同时检测。
