SENSITIVE DETECTION OF ROSS-RIVER-VIRUS - A ONE-TUBE NESTED RT-PCR

A sensitive nested RT-PCR that can be carried out in a single tube is described. The sensitivity of this system was determined, and compared to that of a single round of PCR, and a single round of PCR followed by hybridisation with a radiolabeled oligonucleotide probe. We found t hat with the one-tube nested RT-PCR we were able to detect 0.1 pfu/ml of Ross River virus. The nested RT-PCR was 100-times more sensitive th an a single round of RT-PCR followed by hybridisation, and 10000-times more sensitive than a single round of RT-PCR alone. This system provi des a sensitive detection of Ross River virus, and can be adapted for detection of RNA from any source. The test material is added to a sing le tube at the outset, and by subsequent addition of two sets of reage nts, the entire nested RT-PCR can be carried out in the same tube. Thi s system has maximum sensitivity, minimises risk of contamination, and is amenable to automation.