DEVELOPMENT OF A FAST AMPLIFYRP ACCELER8 DIAGNOSTIC ASSAY FOR GRAPEVINE RED BLOTCH VIRUS

Abstract

Polymerase chain reaction (PCR) is routinely used for the detection of grapevine red blotch virus (GRBV), a virus with a circular single-stranded DNA genome, in grapevine tissue. Since the preparation of purified grapevine DNA is time consuming, a user-friendly AmplifyRP Acceler8 assay was developed to quickly, specifically, and sensitively identify GRBV-infected grapevine samples. The sensitivity of AmplifyRP Acceler8 for GRBV detection is approximately 100 times higher than that of PCR. GRBV is consistently detected by AmplifyRP Acceler8 up to a 10-8 dilution of infected grapevine leaf crude extracts diluted in healthy grapevine leaf crude extracts and nearly 10 copies of plasmid DNA containing a GRBV genomic fragment in a matrix of healthy grapevine leaf crude extracts. The test has no cross reactivity to grapevine tissue, nor to several grapevine-infecting pathogens, including arabis mosaic virus, grapevine fanleaf virus, grapevine fleck virus, grapevine leafroll-associated virus 1, grapevine leafroll-associated virus 2, grapevine leafroll-associated virus 3, grapevine leafroll-associated virus 4 strain 5, tomato 2 ringspot virus, tobacco ringspot virus, Xylella fastidiosa, and Botrytis cinerea. Dried GRBV reaction pellets provided in the AmplifyRP Acceler8 kit contain all the necessary reaction components and are stable for at least 5 weeks at -20°C, 4°C, 22°C, and 37°C, providing convenience for transportation and field application. The AmplifyRP Acceler8 assay generated results consistent with PCR and real-time PCR outputs.