A spot nested RT-PCR method for the simultaneous detection of members of the Vitivirus and Foveavirus genera in grapevine.
A reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to allow rapid, and simultaneous detection of Vitivirus and Foveavirus sequences in two steps. The method involved a one-step RT-PCR, in which the combination of degenerate deoxyinosine-substituted primers amplified part of the polymerase region of both genera, followed by a nested PCR amplification that increased specificity and sensitivity of detection. The increase in sensitivity also permitted the use of a simple and rapid template preparation protocol, involving the spotting of plant sap extract on a nylon membrane. Consistent amplification with infected grapevine plants was possible after inclusion of additives for inhibiting polyphenolic compounds during template preparation. This spot nested RT-PCR method can reliably detect virus species of both genera in grapevine allowing simple, fast, and cost-effective analysis of a large number of samples in certification schemes.
Dovas
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CI
Plant Pathology Laboratory, Faculty of Agriculture, Aristotle University of Thessaloniki, 54 124 Thessaloniki, Greece. dovas@agro.auth.gr
Katis
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NI
D010942:Plant Viruses / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D027843:Vitis / Q000821:virology*