Detection of potato leafroll virus in dormant potato tubers by immunocapture and a fluorogenic 5' nuclease RT-PCR assay

A gelfree, reverse-transcription polymerase chain reaction-based fluorogenic detection method for potato leafroll virus (PLRV) in dormant potato tubers has been designed. A PLRV sequence-specific oligonucleotide (TaqMan probe, Roche Molecular Systems, Inc., Roche Molecular Systems, Inc., Alameda, CA), containing a 5'-terminal 'reporter' fluorescein and a 3'-terminal rhodamine 'quencher,' was specifically degraded during amplification, resulting in a relative increase in reporter-associated fluorescence. The system was coupled to immunocapture of PLRV from tuber sap by paramagnetic beads coated with an antiserum against the virus. Addition of cell wall-degrading enzymes, cellulase, and macerozyme to tuber sap was essential to make detection of PLRV possible on tubers after a storage period. The potential for application in routine detection of PLRV in seed potatoes was investigated by testing the method on primarily infected, dormant tubers of four potato cultivars. Results were validated both by gel electrophoresis and enzyme-linked immunosorbent assay of shoots from sprouted tubers, which is the current assay for postharvest control of PLRV. All methods showed complete agreement in discriminating between PLRV-infected and noninfected samples. Detection took place in microtiter plates and was rapid, reproducible, semi-quantitative, and amenable to automation. The system offers the possibility of reducing the inspection time of seed potatoes for PLRV infection from 5 weeks to 1 day.