VIEW ARTICLE

Techniques

Construction and Use of Cloned cDNA Biotin and ³²P-Labeled Probes for the Detection of Papaya Mosaic Potexvirus RNA in Plants. B. P. Roy, Postdoctorate, Department of Botany, University of Toronto, Toronto, Ontario, Canada, M5S 1A1; M. G. AbouHaidar(2), T. L. Sit(3), and A. Alexander(4). (2)(3)(4)Associate professor, graduate student, and associate professor, Department of Botany, University of Toronto, Toronto, Ontario, Canada, M5S 1A1, (4)Present address: Departement de Biologie, Université de Moncton, New Brunswick, Canada. Phytopathology 78:1425-1429. Accepted for publication 12 June 1988. Copyright 1988 The American Phytopathological Society. DOI: 10.1094/Phyto-78-1425.

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 biotin-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).