Sensitive detection of grapevine virus A, B, or leafroll-associated III from viruliferous mealybugs and infected tissue by cDNA amplification

Abstract

DNA primers specific for grapevine virus A (GVA), grapevine virus B (GVB) or grapevine leafroll-associated virus III (GLRaV-III) were constructed based on the nucleotide sequence of a segment of each viral genome. DNA primers were utilized for cDNA synthesis and polymerase chain reaction (PCR) amplification of a 430 bp fragment from extracts of GVA-infected grapevine tissue or viruliferous mealybugs and 450 bp and 340 bp DNA fragments from extracts of GVB and GLRaV-III-infected grapevine tissues, respectively. The amplified DNA fragment of each virus was identified by Southern hybridization analysis with a cRNA probe of cloned viral genome. Reverse transcription (RT)-PCR, immunocapture (IC)-RT-PCR and/or multiplex (M)-RT-PCR assays were developed for the detection of GVA, GVB, and/or GLRaV-III in extracts of infected grapevine leaves, dormant cuttings and/or in viruliferous mealybugs. Viral specific DNA was absent from amplified extracts of uninfected grapevine tissue or nonviruliferious mealybugs. IC-RT-PCR was easier to perform than RT-PCR for the detection of GVA from viruliferous mealybugs. M-RT-PCR was easier and faster than IC-RT-PCR for the detection of GLRaV-III from infected grapevine tissue and it allows the sensitive detection of GVB, for which a high titer antiserum is not yet available.