Evaluation of a simple membrane-based nucleic acid preparation protocol for RT-PCR detection of potato viruses from aphid and plant tissues.
A rapid and simple protocol for preparing viral RNA from aphids (Myzus persicae) and potato (Solanum tuberosum L.) tissue (leaves, sprouts, and tubers) for reverse transcription-polymerase chain reaction (RT-PCR) was developed. The four-step method involves: (1) preparing plant crude sap or aphid macerates in a buffered detergent (Triton XL-80N) solution; (2) immobilizing clarified sap on a nitrocellulose membrane; (3) performing reverse transcription using eluted water extract from cut-out spot from membrane; and (4) amplifying cDNA through PCR. The entire procedure from tissue grinding to RT-PCR can be completed within 6h. The protocol was applied successfully for the detection of individual potato viruses: carlavirus Potato virus S (PVS), potexvirus Potato virus X (PVX), potyvirus Potato virus Y (PVY), and polerovirus Potato leafroll virus (PLRV). PLRV was also detected from individual viruliferous aphids or composites of viruliferous and healthy aphids. PVY and PLRV were detected from extracts immobilized on nitrocellulose membranes, stored for more than 65-273 days at room temperature (25 degrees C). The protocol was companed with the 'long protocols' involving enzyme and phenol extraction for aphids or sodium sulfite and phenol extraction for tubers. The simplified protocol was found comparable in sensitivity to these long procedures, and is especially suitable for those regions where specialized PCR laboratories are only available in centralized locations. Viral RNA immobilized membranes can be mailed out for detection by RT-PCR to these centralized laboratories from remote areas of the country.
Singh
Rudra P
RP
Agriculture and Agri-Food Canada, Potato Research Centre, PO Box 20280, Fredericton, NB, Canada E3B 4Z7. singhr@agr.gc.ca
Dilworth
Andrea D
AD
Singh
Mathuresh
M
McLaren
Debra L
DL
D000818:Animals; D001042:Aphids / Q000821:virology*; D002021:Buffers; D017788:Carlavirus / Q000235:genetics / Q000302:isolation & purification*; D017793:Luteovirus / Q000235:genetics / Q000302:isolation & purification*; D008567:Membranes, Artificial; D018515:Plant Leaves / Q000821:virology; D035281:Plant Tubers / Q000821:virology; D011092:Polyethylene Glycols; D017863:Potexvirus / Q000235:genetics / Q000302:isolation & purification*; D017800:Potyvirus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D011198:Solanum tuberosum / Q000821:virology*; D013048:Specimen Handling; D013696:Temperature; D013997:Time Factors; D014867:Water
D002021:Buffers; D008567:Membranes, Artificial; D012367:RNA, Viral; D014867:Water; D011092:Polyethylene Glycols