A solvent-free, rapid and simple virus RNA-release method for potato leafroll virus detection in aphids and plants by reverse transcription polymerase chain reaction

A one-step, rapid and economical method for potato leafroll virus (PLRV) RN A release that is applicable to the use on a microcentrifuge scale is descr ibed. Discs (3-6 mm diameter) from leaves, petioles, stems, and tubers of p otato plants were incubated in microcentrifuge tubes with detergent solutio n. The supernatants were used directly for reverse transcription (RT) and p olymerase chain reaction (PCR). Of the seven nonionic detergents of the Tri tonX series evaluated, Triton X405R was the most effective, although X-405 and X-100R were also effective in releasing PLRV RNA. Application of the de tergent method for detecting PLRV in greenhouse-grown potato organs (leaves , petioles, stems, tubers) and in field-grown tubers was demonstrated and c ompared to the multi-step phenol method. When individual aphids, Myzus pers icae, were ground in 20 mu l of detergent solution and supernatants were us ed for RT-PCR, virus was detected in single aphids in undiluted solutions a nd up to a dilution of 1.4. The concentration of PLRV RNA released by the d etergent method was substantially lower than that released by the phenol me thod. However, the detergent method was sensitive enough to detect PLRV fro m potato leaves, petioles, and stems 2 weeks after graft inoculation. The d etergent method was rapid and economical, and has potential for large-scale application. The extracts survived over 37 days at room temperature, thus making it possible to mail extracts from remote areas lacking specialised R T-PCR facilities to a central laboratory for PLRV testing. (C) 1999 Elsevie r Science B.V. All rights reserved.