VIEW ARTICLE

Techniques

Use of a ³²P-Labeled Transcribed RNA Probe for Dot Hybridization Detection of Plum Pox Virus. Christina Varveri, Station de Pathologie Végétale, Institut National de la Recherche Agronomique, Centre de Bordeaux, B.P. 131, 33140 Pont de la Maye, France; T. Candresse, Maria Cugusi, M. Ravelonandro, and J. Dunez. Station de Pathologie Végétale, Institut National de la Recherche Agronomique, Centre de Bordeaux, B.P. 131, 33140 Pont de la Maye, France. Phytopathology 78:1280-1283. Accepted for publication 18 April 1988. Copyright 1988 The American Phytopathological Society. DOI: 10.1094/Phyto-78-1280.

Subcloning of an 800-bp TaqI fragment derived from the 3’ region of cDNA clone of plum pox virus (strain D) RNA in the AccI site of the transcription vector plasmid Bluescribe (pBS⁺) generated plasmid pBPPV1. This plasmid served as a template for phage T₇ RNA polymerase to synthesize ³²P-labeled complementary RNA (cRNA) copies of the viral RNA, which were used as probes in dot blot hybridization assays. A sensitivity of 4 pg of purified virus (PPV-D) per spot was attained with this type of probe. When the above sensitivity was compared with that of a similar nick-translated DNA probe (100 pg virus/spot) or of enzyme-linked immunosorbent assay (ELISA) (1 ng virus/assay), the RNA probe was, respectively, 25 and 250 times more sensitive. We have compared dot-hybridization with an RNA probe to ELISA in routine indexing of infected apricot trees. Almost 100% of positive samples in ELISA were also positive in hybridization, while 76% of samples negative in ELISA indexed positive by hybridization.