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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.

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 6 h. 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 °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.

Polyphenolics are a widely distributed class of plant constituents implicated in the inhibition of the polymerase chain reaction (PCR). Since chlorogenic acid forms the bulk of phenolics in the potato tuber, a range of chlorogenic acid concentrations were tested for inhibition of reverse transcription (RT) and PCR steps. The RT step was inhibited at chlorogenic acid concentrations of 6.0 to 7.5 microg, while the PCR step was inhibited at much lower concentrations of 1.2 to 1.8 microg. Incorporation of 1.2% citric acid at the nucleic acid extraction step prevented darkening of the extract and neutralized the inhibitory effect of chlorogenic acid.

A reverse transcription polymerase chain reaction (RT-PCR) protocol was developed using two 20-mer primers located in nuclear inclusion genes NIa and NIb of potato virus Y (PVY). A 1017 bp PCR-product was detected in dormant potato tubers, infected with PVY O , but not in tubers from healthy plants. The PCR product was specific to PVY, as determined by Southern blot detection by hybridization with a PVY O -specific probe. As little as 1 pg of purified PVY O -RNA can be detected after RT-PCR amplification. The presence of phenolics or polysaccharides in tuber nucleic acids inhibited PVY O amplification, which was eliminated by diluting nucleic acid preparations prior to cDNA synthesis, modifying the nucleic acid extraction procedure by isopropanol precipitation and using phosphate-buffered saline-Tween in the cDNA mix. Potato cultivars differed in PVY O concentration in tubers as much as 128-fold. Tuber parts used for nucleic acid extractions were important in potato cultivars with low virus titres and combining of saps from bud and stem ends improved the PVY O detection. Storage of tubers up to 90 days at 4 °C did not result in reduced detection of PVY O by both nucleic acid spot hybridization and RT-PCR, but RT-PCR band intensity was lower at longer storage periods. The primer pair developed in this study exhibited broad specificities with field isolates from Peru, Scotland and North America.

An isolate (Y139) of the potato virus Y strain PVYO, common in the potato-growing regions of eastern Canada, was cloned and segments of about 1800 bases from the 3'-end of the PVY-genome were sequenced. These clones were used to prepare molecular detection probes by labelling with digoxigenin. Five methods reported to yield nucleic acid preparations from tuber tissue were compared. Two of the methods failed to yield nucleic acids from tubers suitable for use in dot-blot hybridization. The other three methods yielded satisfactory nucleic acids which could be successfully detected with the PVYO probes. A method of nucleic acid extraction from tuber tissues was selected which is simple, reproducible and does not use phenol. Using this standardized procedure, PVY was detected in freshly harvested, greenhouse-grown tubers of 8 potato cultivars; the probe was specific to PVY; and the sensitivity of detection using purified viral RNA was down to 10 pg. The sensitivity of detection in tuber extracts was down to a dilution of 1:64, while that from potato leaves was to a dilution of 1:1024. Nucleic acids prepared by the standardized procedure and denatured prior to storage at 4 degrees C, -20 degrees C or at -70 degrees C remained suitable for hybridization for over 2 weeks.

Test conditions for the simultaneous detection of potato leafroll virus (PLRV) and potato virus Y (PVY) in dormant tubers and leaves by reverse transcription-polymerase chain reaction (RT-PCR) were optimized. Various factors optimized at the reverse transcription (RT) stage rather than at the amplification (PCR) stage affected the outcome. In the simplex RT-PCR a onefold dNTPs concentration (0.5 mM) was sufficient in yielding a PLRV or PVY band. In contrast, the duplex RT-PCR required a minimum twofold dNTPs concentration (1.0 mM) during RT to produce distinct bands in PCR. Similarly, various proportions of antisense primers of PLRV and PVY used during RT affected subsequent duplex RT-PCR. Optimal amplification of both viruses were obtained at a ratio of 0.90:0.49 microM of PLRV:PVY antisense primers. An interaction of dNTPs and RNA template concentration was observed. A higher concentration of RNA was required at onefold dNTPs concentration than at twofold dNTPs. Dilutions down to 1:300 of RNA template yielded distinct bands of both viruses at twofold dNTPs concentration. At optimized conditions of the duplex RT-PCR both viruses were reliably detected in composite samples at a ratio of one part infected sap mixed with 399 parts of sap from healthy tubers. Application of optimized conditions to singly- and doubly-infected tubers detected both viruses from naturally infected field-grown tubers. A nearly perfect correlation (r(2)=0.99) was observed between visible plant symptoms and the virus detection from leaves and tubers by the duplex RT-PCR.