Development of a fluorogenic RT-PCR assay (TaqMan) for the detection of Hendra virus

Abstract

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.