Detection of different strains of Potato virus Y and their mixed infections using competitive fluorescent RT-PCR

A competitive fluorescent RT-PCR assay (CF RT-PCR) was developed for the ra pid and reliable detection and discrimination of the two most common strain s of Potato virus Y (PVY) found in potato (necrotic and ordinary). The assa y incorporates two strain specific primers labelled with fluorescent labels , used in conjunction with a universal PVY primer. The strain specific prim ers compete for the same annealing site which further increases specificity . Discrimination is conferred by the fluorescent labels; green PCR products for PVYO and red for PVYN, whilst mixed infections are detected as orange PCR products without the need for staining agarose gels. The assay can be s caled up for the processing of 96 samples simultaneously, with the detectio n of PCR products directly using a fluorescent microtitre plate reader. The assay successfully discriminated between 20 isolates of PVY tested, and co uld be used for the direct detection of PVY in potato tubers. (C) 2001 Else vier Science B.V. All rights reserved.