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Techniques

Improved Detection of Potato Leafroll Virus in Plant Material and in Aphids. J. F. J. M. van den Heuvel, Department of Virology, Wageningen Agricultural University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands; D. Peters, Department of Virology, Wageningen Agricultural University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands. Phytopathology 79:963-967. Accepted for publication 10 February 1989. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1989. DOI: 10.1094/Phyto-79-963.

To improve detection of potato leafroll virus (PLRV) in plant material and viruliferous aphids, the enzyme-linked immunosorbent assay (ELISA) was modified by simultaneous incubation of sample and conjugate (cocktail ELISA) and by amplification of the enzyme reaction. Absorbance values of PLRV-containing samples in the cocktail ELISA were higher than those of comparable samples in a sandwich ELISA procedure in which sample and conjugate were incubated sequentially. Addition of sodium diethyldithiocarbamate or ethylenediaminetetraacetic acid to the sample buffer in the cocktail ELISA significantly increased the absorbance values of infected plant material, while the background signals were reduced. Amplification of the enzyme reaction, in which dephosphorylated substrate catalytically triggered an enzyme-mediated redox cycle, further increased sensitivity. Using this technique, 50–100 pg of PLRV could be detected per sample, and the virus could be detected in highly diluted leaf sap and in single Myzus persicae nymphs after a 12-hr acquisition access period on PLRV-infected Physalis floridana plants. In addition, when coated plates were used, the total assay time for PLRV detection in plant material could be reduced to 20 min.