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Egypt. J. Exp. Biol. (Bot.). 2008; 4(0): 71-78


IMPROVED TECHNIQUES FOR THE PURIFICATION AND SEROLOGY OF AN EGYPTIAN ISOLATE OF ONION YELLOW DWARF VIRUS (OYDV).

Ibrahim L. Massoud Ahmed A. Abo-Elela.

Abstract
Three different procedures of purification were performed to purify a local isolate of Onion yellow dwarf potyvirus (OYDV). Two of them resulted in a low yield of aggregated virions. Improved technique was devised to overcome the problem of aggregation of OYDV particles. The inclusion of stabilizing additives in the extraction and re-suspending buffers proved very useful in preventing virus aggregation. The purified virus formed a single opalescent zone in density gradient columns. The absorption spectrum of the purified virus was typical for nucleoproteins with maximum and minimum absorbance values at 260 and 245 nm. The A 260/280, A 280/260 and A Max/Min ratios were 1.201, 0.831, and 1.102, respectively. Electron microscopy of purified virus preparation showed filamentous flexuous particles with 750-800 nm length. The polyclonal antibodies were raised against the locally purified OYDV isolate. The titer as determined by indirect ELISA was 1/4096. ELISA-Kit was produced and successfully used for virus detection by using the optimum concentration of 1 ug/ml and 1:1000 for IgG and IgG- conjugate, respectively. It was found that the proper time for detecting OYDV is three months after transplanting onion plants grown in the field. Best samples displaying high concentration of OYDV were those taken from the middle leaves of the plant. Some serological tests were developed for virus detection in leaves and seeds such as ELISA. dot-ELISA and direct tissue blotting. Improving the used techniques was important for increasing the sensitivity of the reaction, minimizing its time and reducing the cost.

Key words: Onion yellow dwarf virus, antigen, antiserum, ELISA-Kit, electron microscopy, dot-ELISA and direct- tissue blotting.



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