Quantitative determination of tomato yellow leaf curl geminivirus DNA by chemiluminescent assay using digoxigenin-labeled probes.
A quantitative dot-blot hybridisation assay was developed for tomato yellow leaf curl geminivirus (TYLCV). The assay is based on chemiluminescent detection of viral and plasmid DNA using digoxigenin-labeled probes on nylon membranes. The response-error relationship was studied and a square root transformation was found to stabilise the variance of the response. An asymmetric sigmoid (gompertz) curve was used to describe the dose-response relationship. The detection limits and the precision profiles of the curves were studied. A method is suggested for setting an upper limit on the maximum DNA amount that can be discriminated from the upper asymptote. With respect to different times of exposure of an X-ray film to a membrane, the shortest times gave better upper limits and the longest times provided better detection limits. Purified virus and plasmid preparations were studied in various dilution media, such as TYLCV-free Bemisia tabaci (the whitefly vector) and tomato extracts, with particular attention to parallelism with standard calibration curves. Plasmid diluted in buffer was found useful for calibration of purified virus and virus in the vector, while extraction of known amounts of virus, in parallel with samples to be examined, was needed to quantify viral DNA in plant hosts.
Caciagli
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P
Istituto di Fitovirologia Applicata, Torino, Italy.
Bosco
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D
D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis*; D004076:Digoxigenin; D017791:Geminiviridae / Q000235:genetics*; D008163:Luminescent Measurements; D015233:Models, Statistical; D009693:Nucleic Acid Hybridization
D015342:DNA Probes; D004279:DNA, Viral; D004076:Digoxigenin