QUANTITATIVE-DETERMINATION OF TOMATO YELLOW LEAF CURL GEMINIVIRUS DNABY CHEMILUMINESCENT ASSAY USING DIGOXIGENIN-LABELED PROBES

A quantitative dot-blot hybridisation assay was developed for tomato y ellow leaf curl geminivirus (TYLCV). The assay is based on chemilumine scent detection of viral and plasmid DNA using digoxigenin-labeled pro bes on nylon membranes. The response-error relationship was studied an d a square root transformation was found to stabilise the variance of the response. An asymmetric sigmoid (gompertz) curve was used to descr ibe the dose-response relationship. The detection limits and the preci sion profiles of the curves were studied. A method is suggested for se tting an upper limit on the maximum DNA amount that can be discriminat ed from the upper asymptote. With respect to different times of exposu re of an X-ray from to a membrane, the shortest times gave better uppe r limits and the longest times provided better detection limits. Purif ied virus and plasmid preparations were studied in various dilution me dia, such as TYLCV-free Bemisia tabaci (the whitefly vector) and tomat o extracts, with particular attention to parallelism with standard cal ibration curves. Plasmid diluted in buffer was found useful for calibr ation of purified virus and virus in the vector, while extraction of k nown amounts of virus, in parallel with samples to be examined, was ne eded to quantify viral DNA in plant hosts.