An alkaline solution simplifies nucleic acid preparation for RT-PCR and infectivity assays of viroids from crude sap and spotted membrane.
Formats of a simple protocol for the preparation of nucleic acids for infectivity and RT-PCR detection of viroids from minute amounts of plant material are described. The method consists of preparing crude extracts in a NaOH-EDTA solution and then testing the supernatant. The NaOH-EDTA extract can be used at four distinct stages of preparation depending upon the accuracy desired, namely: (1) incubation of extract for 15 min at room temperature and the use of the supernatant for RT-PCR; (2) the supernatant can be spotted onto a nitrocellulose membrane (NCM) without vacuum, and the water-eluted liquid is used for RT-PCR; (3) centrifugation of the extract and use of supernatant in RT-PCR; (4) for quantitative accuracy, spotting the centrifuged supernatant on NCM using a vacuum device and then using the water-eluted liquid for RT-PCR. The protocols are rapid, inexpensive and applicable to large-scale epidemiological survey of ornamental plants or crops. The membranes are easily transported long distances and can be stored at room temperature for several months while retaining the ability to detect viroids by RT-PCR and by infectivity assays.
Singh
Rudra P
RP
Potato Research Centre, Agriculture and Agri-Food Canada, 850 Lincoln Road, P.O. Box 20280, Fredericton, New Brunswick, Canada E3B 4Z7. singhr@agr.gc.ca
Dilworth
Andrea D
AD
Singh
Mathuresh
M
Babcock
Krista M
KM
D000327:Adsorption; D003101:Collodion / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D018551:Solanum lycopersicum / Q000821:virology*; D008851:Micropore Filters; D010935:Plant Diseases / Q000821:virology*; D018515:Plant Leaves / Q000821:virology; D035281:Plant Tubers / Q000821:virology; D012367:RNA, Viral / Q000302:isolation & purification* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D011198:Solanum tuberosum / Q000821:virology*; D014772:Viroids / Q000235:genetics* / Q000302:isolation & purification*
D012367:RNA, Viral; D003101:Collodion