Smith I L, Halpin K, Warrilow D, Smith G A
Public Health Virology, Queensland Health Scientific Services, 39 Kessels Road, Queensland 4108, Coopers Plains, Australia.
J Virol Methods. 2001 Oct;98(1):33-40. doi: 10.1016/s0166-0934(01)00354-8.
A rapid and sensitive one-tube RT-PCR assay using a fluorogenic (TaqMan) probe was developed to improve the diagnosis of Hendra virus (HeV) infection. The TaqMan assay was developed to rapidly and specifically identify Hendra virus. The sensitivity of the new TaqMan-based PCR assay compared favourably with conventional RT-PCR. The major advantage of the TaqMan-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.
为了改进亨德拉病毒（HeV）感染的诊断，开发了一种使用荧光（TaqMan）探针的快速灵敏的单管逆转录聚合酶链反应（RT-PCR）检测方法。开发TaqMan检测方法是为了快速、特异性地鉴定亨德拉病毒。基于TaqMan的新型PCR检测方法的灵敏度优于传统RT-PCR。基于TaqMan的检测方法的主要优点是诊断速度快，在完成PCR后几分钟内即可得到结果，在收到标本后4小时内即可得到结果。该检测方法通过省去第二轮PCR和对琼脂糖凝胶的需求，大大降低了假阳性的几率。设计、克隆并体外转录了重组引物对照（由啮齿动物甘油醛-3-磷酸脱氢酶（GADPH）探针序列两侧的亨德拉病毒引物序列组成）和重组探针对照（由亨德拉病毒探针序列两侧的啮齿动物GADPH引物序列组成）以产生RNA。这减少了将病毒RNA用作阳性对照的需求，从而降低了产生假阳性的几率，同时消除了与处理活病毒相关的生物安全风险。该检测方法将为未来亨德拉病毒的爆发提供快速诊断。
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