Use of [32P]RNA probes for the dot-hybridization detection of potato spindle tuber viroid

A comparative study of the sensitivity of dot hybridization assays using cRNA and cDNA for the detection of potato spindle tuber viroid.

“…This means that the detection sensitivity of the cRNA probe is eight to 64 times higher than that of the cDNA, using both probes at a final radioactivity of 2.0X10° cpm/ml. Previously, Lakshman et al (1986) reported that the use of the cRNA probe (0.7x10° cpm/ml) detected as little as probes from pAV 401 (0.7X 10G cpm/ml) or from the 358 by PSTV-specific DNA (0.5X 106 cpm!ml) detected 45 pg or 11.2 pg of purified PSTV in clarified sap from uninoculated tomato leaves and PSTV in a 1:5 dilution of infected tomato sap. Thus, the differences in the sensitivity limits of the cRNA probe were four to 32 times higher than those of the cDNA probe for purified PSTV and five times higher for sap samples (Lakshman et al, 1986 In a range of 10 to 30°C, potato plants kept at 25°C yielded the highest amount of PSTV as detected by both the cRNA and cDNA probes.…”

“…Previously, Lakshman et al (1986) reported that the use of the cRNA probe (0.7x10° cpm/ml) detected as little as probes from pAV 401 (0.7X 10G cpm/ml) or from the 358 by PSTV-specific DNA (0.5X 106 cpm!ml) detected 45 pg or 11.2 pg of purified PSTV in clarified sap from uninoculated tomato leaves and PSTV in a 1:5 dilution of infected tomato sap. Thus, the differences in the sensitivity limits of the cRNA probe were four to 32 times higher than those of the cDNA probe for purified PSTV and five times higher for sap samples (Lakshman et al, 1986 In a range of 10 to 30°C, potato plants kept at 25°C yielded the highest amount of PSTV as detected by both the cRNA and cDNA probes. This result is not necessarily in agreement with the previous reports by Harris and Browning (1980) and Morris and Smith (1977) who reported that viroid concentration increased faster at 30-31°C than at 23-25°C.…”

“…Since its first application to the detection of potato spindle tuber viroid (PSTV) by Owens and Diener (1981), the DNA dot hybridization technique has been the subject of active investigations, and several groups have contributed to simplification of the procedures and improvement of sensitivity limits (Macquaire et al, 1984;Bar-Joseph et al, 1985;Barker et al, 1985;Lakshman et al, 1986;Bernardy et al, 1987). With the use of SP6 cloning vectors it is possible to transcribe inserted DNA into RNA probes of high specific activity in vitro (Melton et al, 1984).…”

“…The procedure for PSTV purification was essentially the same as described previously (Lakshman et al, 1986). Potato plants were inoculated with a sterile cotton swab dipped in PSTV-inoculum (5 µg/ml), when they were about 5 to 7 cm high.…”

“…The plasmids were propagated in Escherichia toll strain HB101 in the presence of ampicillin, and purified by equilibrium centrifugation in a CsCI-EtBr gradient (Clewell and Helinski, 1972). The recombinant plasmid containing PSTV-specific DNA insert pDX4, obtained in the previous study (Lakshman et al, 1986), was used for preparing 32P-labelled RNA probe.…”