VIEW ARTICLE

Techniques

Comparison of Dot Molecular Hybridization and Enzyme-Linked Immunosorbent Assay for Detecting Tobacco Mosaic Virus in Plant Tissues and Protoplasts. Ilan Sela, Professor, The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot 76100 Israel; Michal Reichman(2), and Arthur Weissbach(3). (2)Graduate student, The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot 76100 Israel; (3)Associate director, Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110. Phytopathology 74:385-389. Accepted for publication 31 August 1983. Copyright 1984 The American Phytopathological Society. DOI: 10.1094/Phyto-74-385.

A randomly ³²P-labeled DNA probe was prepared by reverse-transcribing tobacco mosaic virus (TMV) RNA to produce single-stranded complementary DNA (cDNA). A dot hybridization technique using a (³²P)cDNA probe was adapted for detecting TMV-RNA in crude leaf sap and from protoplasts derived from cultured cells. Tissue homogenates or lysed protoplasts were briefly denatured and applied directly to a nitrocellulose membrane without further clarification. As little as 2.5 pg of purified TMV-RNA can be detected. The rate of synthesis of TMV-RNA as measured by the dot hybridization technique was similar to that obtained with the enzyme-linked immunosorbent assay (ELISA) for the appearance of the TMV capsid protein. However, the dot hybridization method is about twice as sensitive and detects the appearance of new TMV-RNA earlier in infection than the ELISA method which only detects new capsid protein.