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) RNA release that is applicable to the use on a microcentrifuge scale is described. Discs (3-6 mm diameter) from leaves, petioles, stems, and tubers of potato plants were incubated in microcentrifuge tubes with detergent solution. The supernatants were used directly for reverse transcription (RT) and polymerase chain reaction (PCR). Of the seven nonionic detergents of the TritonX series evaluated, Triton X405R was the most effective, although X-405 and X-1OOR were also effective in releasing PLRV RNA. Application of the detergent method for detecting PLRV in greenhouse-grown potato organs (leaves, petioles, stems, tubers) and in field-grown tubers was demonstrated and compared to the multi-step phenol method. When individual aphids, Myzus persicae, were ground in 20 microl of detergent solution and supernatants were used for RT-PCR, virus was detected in single aphids in undiluted solutions and up to a dilution of 1:4. The concentration of PLRV RNA released by the detergent method was substantially lower than that released by the phenol method. However, the detergent method was sensitive enough to detect PLRV from potato leaves, petioles, and stems 2 weeks after graft inoculation. The detergent 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 RT-PCR facilities to a central laboratory for PLRV testing.
Singh
R P
RP
Agriculture and Agri-Food Canada, Potato Research Centre, Fredericton, NB. singhr@em.agr.ca
D000818:Animals; D001042:Aphids / Q000821:virology; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D003902:Detergents; D017793:Luteovirus / Q000235:genetics* / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D011198:Solanum tuberosum / Q000821:virology; D012997:Solvents; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D003902:Detergents; D012367:RNA, Viral; D012997:Solvents; D011092:Polyethylene Glycols