A one-tube, one manipulation RT-PCR reaction for detection of Ross River virus.
A sensitive, single tube reverse transcription-polymerase chain reaction (RT-PCR) protocol for the detection of Ross River virus (RRV) is described. All components necessary for both reverse transcription and PCR were combined in a single tube, and reverse transcription and PCR carried out sequentially in a single, non-interrupted thermal cycling program. The antisense oligonucleotide from the two primers selected for use in the PCR also served to prime specifically for the reverse transcription. The 549 bp product was detected by electrophoresis and ethidium bromide staining. The detection limit using this system was 18 fg of purified viral RNA or 1.3 pfu of whole virus. Greater sensitivity cannot reasonably be expected unless a more sensitive method than electrophoresis and ethidium bromide staining is used for PCR product detection, such as nested PCR or hybridisation with labelled probe. This PCR detection system will be adapted for detection of RRV in mosquito populations for virus surveillance programs.
U.W.A. Department Microbiology, QEII Medical Centre, Perth, Australia.
L N
LN
Sellner
R J
RJ
Coelen
J S
JS
Mackenzie
D000818:Animals; D001483:Base Sequence; D009033:Culicidae / Q000382:microbiology; D004279:DNA, Viral / Q000235:genetics; D005069:Evaluation Studies as Topic; D006801:Humans; D007303:Insect Vectors / Q000382:microbiology; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012398:Ross River virus / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D010948:Viral Plaque Assay; D014773:Virology / Q000379:methods*; D014776:Virus Cultivation
D004279:DNA, Viral; D015345:Oligonucleotide Probes; D012367:RNA, Viral