PCR amplification of a specific double-stranded RNA region of Fiji disease virus from diseased sugarcane.
A 450-bp region from one species of the segmented dsRNA genome of Fiji disease virus (FDV) was amplified from total nucleic acid extracts of diseased plants by reverse transcription with MMLV, followed by amplification with Taq DNA polymerase (RT-PCR). Other FDV-specific regions (c 150 bp and c 270 bp) were also amplified from the dsRNA template. FDV cDNA was only synthesised when the viral dsRNA template was boiled and quenched with FDV-specific or random hexamer primers. The reverse transcriptase/DNA polymerase enzyme rTth appeared to yield only the 150 bp fragment from the dsRNA template under the conditions used. The level of sensitivity of RT-PCR for purified FDV dsRNA was 100 ag, approximately 10(4)-fold more sensitive than detection with biotinylated DNA probe.
Smith
G R
GR
Bureau of Sugar Experiment Stations, Indooroopilly, Queensland, Australia.
Van de Velde
R
R
Dale
J L
JL
D001483:Base Sequence; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000235:genetics* / Q000382:microbiology*; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D015347:RNA Probes; D012330:RNA, Double-Stranded / Q000302:isolation & purification*; D012367:RNA, Viral / Q000302:isolation & purification*; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity
D015347:RNA Probes; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase