Novel Methods Based on Genomic Amplification for Routine Detection of Potato Leafroll Virus in Potato Tubers

Abstract

Two novel detection methods for potato leafroll virus (PLRV) in potato tubers based on genomic amplification have been developed: a one-tube, fluorogenic 5’ nuclease RT-PCR assay and an isothermal NASBA~ amplification assay. The former method consists of an immunocapture of PLRV coupled to a one-tube RT-PCR using Thermus thermophilus (rTth) instead of Taq DNA polymerase. Fluorescent detection in a microwell format was realized incorporating into the reaction a PLRV-specific probe(TaqMan^TM probe) that carries a 5’- terminal ‘reporter’ fluorescein and a 3’-terminal rhodamine ‘quencher’. During the extension phase of PCR, the probe was specifically degraded by the exonuclease activity of the DNA polymerase, resulting in an increase in reporter fluorescence at each cycle. The NASBA method allowed for amplification of PLRV RNA in a single enzymatic reaction at 41°C by the concurrent activity of AMV reverse transcriptase, T7 RNA polymerase and RNase H. A ten-fold dilution in water of extracts of both a healthy tuber spiked with purified PLRV and a primarily PLRV-infected tuber was sufficient to allow amplification and detection by agarose gel electrophoresis. Both systems offer the possibility to reduce the inspection time of seed-potatoes for infection of PLRV from the current five weeks to one day.