PCR assays for the detection of members of the genus Ilarvirus and family Bromoviridae.
A PCR assay was developed for the universal detection of ilarviruses using primers designed to the RNA-dependent RNA polymerase gene in RNA2. The assay detected 32 isolates of 15 definite and 2 tentative ilarvirus species using a one-step RT-PCR. The assay was more specific, and at least as sensitive as a commercial assay, and allowed direct sequencing of amplicons. No cross-reaction was observed with neither healthy plants of 15 host species nor from isolates in other genera of the Bromoviridae. A further PCR assay targeting the helicase motif of RNA1 was able to detect all species tested within the family Bromoviridae, including members of the Alfamovirus, Anulavirus, Bromovirus, Cucumovirus and Ilarvirus. The assays provide a sensitive and cost-effective way for detecting and characterising members of the Bromoviridae and can be used for quarantine and certification programmes.
Untiveros
Milton
M
Plant Health and Environment Laboratory, Investigation and Diagnostic Centre, MAF Biosecurity New Zealand, PO Box 2095, Auckland 1140, New Zealand.
Perez-Egusquiza
Zoila
Z
Clover
Gerard
G
D019177:Bromoviridae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D003429:Cross Reactions; D017931:DNA Primers / Q000235:genetics; D008969:Molecular Sequence Data; D020365:RNA Helicases / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014764:Viral Proteins / Q000235:genetics
D017931:DNA Primers; D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase; D020365:RNA Helicases