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 radiolabelled oligonucleotide probe. We found that 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 than a single round of RT-PCR followed by hybridisation, and 10,000-times more sensitive than a single round of RT-PCR alone. This system provides 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 single tube at the outset, and by subsequent addition of two sets of reagents, the entire nested RT-PCR can be carried out in the same tube. This system has maximum sensitivity, minimises risk of contamination, and is amenable to automation.
Sellner
L N
LN
Department of Microbiology, QEII Medical Centre, University of Western Australia, Nedlands, Perth.
Coelen
R J
RJ
Mackenzie
J S
JS
D018354:Alphavirus Infections / Q000175:diagnosis; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012398:Ross River virus / Q000235:genetics* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D010948:Viral Plaque Assay; D014773:Virology / Q000379:methods
D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral