DAS-ELISA was carried out as described by Clark and Adams with some minor modifications, using a commercially available PVY IgG and the alkaline phosphatase-conjugated PVY IgG. Polystyrene microtiter plates were coated for 3 h at 34Â°C, with 200 Î¼l per well of IgG coating, in 50 mM carbonate buffer, pH 9.6. The plates were then incubated for 1 h at 34Â°C with PBS (10 mM phosphate buffer, pH 7.2, 0.8% NaCl and 0.02% KCl). After that, the plates were washed three times using washing buffer (0.8% NaCl, pH 7.2 and 0.05% Tween 20). The infection-free (control) and PVY-infected potato leaf samples were ground in ten volumes (w/v) of PBS buffer pH 7.2, containing 0.2% polyvinyl pyrrolidone and 2% of egg albumin (Sigma A5253).The infected preparations were serially diluted (fivefold dilution) at the same buffer. Aliquots of 195 Î¼l of prepared samples were added to each well, and the plates were incubated overnight at 4Â°C. Plates were then washed three times with washing buffer, incubated for 4 h at 37Â°C, with 190 Î¼l per well of alkaline phosphatase-conjugated PVY IgG diluted in sample buffer, washed again, and incubated lastly for 90 min, with p-nitrophenylphosphate (1 mg/ml), in 10% diethanolamine, pH 9.8. Data were expressed and recorded using Multiskan at A405 nm.
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Citation: Almasi MA, Dehabadi SH (2013) Colorimetric Immunocapture Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid Detection of the Potato virus Y. J Plant Pathol Microb 4: 188
Last date updated on July, 2014