Construction and Use of Cloned cDNA Biotin and32P-Labeled Probes for the Detection of Papaya Mosaic Potexvirus RNA in Plants

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Abstract
Complementary DNA to papaya mosaic potexvirus RNA (PMV RNA) was synthesized by reverse transcription, cloned into pUC 18, and amplified in Escherichia coli. RNA probes were prepared by random-primed labeling (using either botin-7-dATP or 32P-dATP) from plasmids containing inserts representing approximately 97% of the viral genome. Partially purified total RNAs from healthy and infected plants were spotted onto nitrocellulose filters and hybridized to biotin- and 32P-labeled probes. Different blocking and stringent washing conditions for cDNA and cloned PMV DNA probes resulted in the reduction of the nonspecific signals with cDNA probes and even total elimination with cloned DNA probes. The minimum detection level of PMV RNA using biotinylated probes made from cloned DNA or complementary DNA to viral RNAs was about 50 pg. Glyoxalation or formylation of total plant RNAs before spotting onto nitrocellulose filters increased by several fold the sensitivity of detection. The detection limit for the biotin-labeled probes was similar to 32P-labeled ones and comparable to the enzyme-linked immunosorbent assay (ELISA).