Development of a multiplex AmpliDet RNA for the simultaneous detection of Potato leafroll virus and Potato virus Y in potato tubers.
A novel isothermal multiplex AmpliDet RNA system is described for the simultaneous amplification and detection of Potato leafroll virus (PLRV) and Potato virus Y (PVY) in seed potatoes. The risk of contamination by carry-over during diagnostic screening is eliminated by performing the reaction in a single closed tube. The viruses present in a sample are identified using differently coloured molecular beacons directed to a selected virus-specific sequence within the amplicon formed during amplification. With this system, as little as 10 fg of purified PLRV or PVY can be detected. The presence of both viruses in a sample is detected by the multiplex assay within a high range of virus concentrations. The reliability of the multiplex assay was compared with the enzyme-linked immunosorbent assay for detection of PLRV- or PVY-antigens in potato tubers. The multiplex assay detected clearly the viruses present originally in the potato tubers in all samples, demonstrating its potential for routine diagnostic work and high-throughput screening.
Klerks
M M
MM
Plant Research International BV, PO Box 16, 6700 AA Wageningen, The Netherlands. m.m.klerks@plant.wag-ur.nl
Leone
G O
GO
Verbeek
M
M
van den Heuvel
J F
JF
Schoen
C D
CD
D003199:Computer Systems; D005456:Fluorescent Dyes; D017793:Luteovirus / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015345:Oligonucleotide Probes; D010935:Plant Diseases / Q000821:virology*; D017800:Potyvirus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis; D012680:Sensitivity and Specificity; D011198:Solanum tuberosum / Q000821:virology*
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012367:RNA, Viral