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 fluor ogenic detection method for potato leafroll virus (PLRV) in dormant po tato tubers has been designed. A PLRV sequence-specific oligonucleotid e (TaqMan probe, Roche Molecular Systems, Inc., Roche Molecular System s, Inc., Alameda, CA), containing a 5'-terminal 'reporter' fluorescein and a 3'-terminal rhodamine 'quencher,' was specifically degraded dur ing amplification, resulting in a relative increase in reporter-associ ated fluorescence. The system was coupled to immunocapture of PLRV fro m tuber sap by paramagnetic beads coated with an antiserum against the virus. Addition of cell wall-degrading enzymes, cellulase, and macero zyme to tuber sap was essential to make detection of PLRV possible on tubers after a storage period. The potential for application in routin e detection of PLRV in seed potatoes was investigated by testing the m ethod on primarily infected, dormant tubers of four potato cultivars. Results were validated both by gel electrophoresis and enzyme-linked i mmunosorbent assay of shoots from sprouted tubers, which is the curren t assay for postharvest control of PLRV. All methods showed complete a greement in discriminating between PLRV-infected and noninfected sampl es. Detection took place in microtiter plates and was rapid, reproduci ble, semi-quantitative, and amenable to automation. The system offers the possibility of reducing the inspection time of seed potatoes for P LRV infection from 5 weeks to 1 day.