Development of a fluorogenic RT-PCR assay (TaqMan) for the detection of Hendra virus
Development of a fluorogenic RT-PCR assay (TaqMan) for the detection of Hendra virus
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DOI：
10.1016/s0166-0934(01)00354-8
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发表时间：
2001-10-01
影响因子：
3.1
通讯作者：
Smith, GA
中科院分区：
文献类型：
--
作者：
Smith, IL;Halpin, K;Smith, GA
为提高亨德拉病毒（HeV）感染的诊断水平，采用荧光TaqMan （TM）探针建立了一种快速、灵敏的单管RT-PCR检测方法。TaqMan （TM）检测是为了快速、特异地鉴定亨德拉病毒而开发的。与传统的RT-PCR相比，新的TaqMan (TM) PCR检测的敏感性更好。TaqMan （TM）检测的主要优点是诊断速度快，在完成PCR的几分钟内和收到标本的4小时内就能得到结果。该测试通过消除第二轮PCR和琼脂糖凝胶的需求，大大减少了假阳性的机会。设计了由亨德拉病毒引物序列侧翼的啮齿动物GADPH探针序列组成的重组引物对照和由亨德拉病毒探针序列侧翼的啮齿动物GADPH引物序列组成的重组引物对照，并进行了克隆和体外转录以生成RNA。这降低了将病毒RNA用作阳性对照的要求，从而减少了产生假阳性的机会，同时消除了与处理活病毒相关的生物安全风险。该试验将提供对未来亨德拉病毒爆发的快速诊断。(C) 2001 Elsevier Science B.V.版权所有
A rapid and sensitive one-tube RT-PCR assay using a fluorogenic (TaqMan (TM)) probe was developed to improve the diagnosis of Hendra virus (HeV) infection. The TaqMan (TM) assay was developed to rapidly and specifically identify Hendra virus. The sensitivity of the new TaqMan (TM) -based PCR assay compared favourably with conventional RT-PCR. The major advantage of the TaqMan (TM) -based assay was the speed of diagnosis with results available within minutes of completing the PCR, and within 4 h of receiving the specimen. This test greatly reduces the chance of false positives through the elimination of second-round PCR and the requirement for agarose gel. Recombinant primer controls consisting of the Hendra virus primer sequence flanking a rodent GADPH probe sequence and recombinant probe controls consisting of the rodent GADPH primer sequence flanking the Hendra virus probe sequence were designed, cloned and transcribed in vitro to generate RNA. This has alleviated the requirement for viral RNA to be used as positive controls, thus reducing the chance of producing a false positive, at the same time eliminating the biosafety risk associated with handling live virus. This assay will provide a rapid diagnosis of future outbreaks of Hendra virus. (C) 2001 Elsevier Science B.V. All rights reserved.