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

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) infec tion. The TaqMan (TM) assay was developed to rapidly and specifically ident ify Hendra virus. The sensitivity of the new TaqMan (TM) -based PCR assay c ompared favourably with conventional RT-PCR. The major advantage of the Taq Man (TM) -based assay was the speed of diagnosis with results available wit hin minutes of completing the PCR, and within 4 h of receiving the specimen . This test greatly reduces the chance of false positives through the elimi nation 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 th e rodent GADPH primer sequence flanking the Hendra virus probe sequence wer e designed, cloned and transcribed in vitro to generate RNA. This has allev iated the requirement for viral RNA to be used as positive controls, thus r educing the chance of producing a false positive, at the same time eliminat ing the biosafety risk associated with handling live virus. This assay will provide a rapid diagnosis of future outbreaks of Hendra virus. (C) 2001 El sevier Science B.V. All rights reserved.