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 rapid and reliable detection and discrimination of the two most common strains of Potato virus Y (PVY) found in potato (necrotic and ordinary). The assay incorporates two strain specific primers labelled with fluorescent labels, used in conjunction with a universal PVY primer. The strain specific primers compete for the same annealing site which further increases specificity. Discrimination is conferred by the fluorescent labels; green PCR products for PVY(O) and red for PVY(N), whilst mixed infections are detected as orange PCR products without the need for staining agarose gels. The assay can be scaled up for the processing of 96 samples simultaneously, with the detection of PCR products directly using a fluorescent microtitre plate reader. The assay successfully discriminated between 20 isolates of PVY tested, and could be used for the direct detection of PVY in potato tubers.
Walsh
K
K
Central Science Laboratory MAFF, Sand Hutton, York, YO41 1LZ, UK. k.walsh@csl.gov.uk
North
J
J
Barker
I
I
Boonham
N
N
D000914:Antibodies, Viral / Q000276:immunology; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes; D010947:Plants, Toxic; D017800:Potyvirus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D011198:Solanum tuberosum / Q000821:virology; D013045:Species Specificity; D014026:Nicotiana
D000914:Antibodies, Viral; D017931:DNA Primers; D005456:Fluorescent Dyes; D012367:RNA, Viral