PETRI DISH ELISA: AN ECONOMIC TECHNIQUE FOR DETECTION OF SEED BORNE VIRUSES
ME ABDALLA1 and SE ALBRECHTSEN2
1Plant Pathology Dept., Faculty of Agriculture, Mansoura University, Egypt; 2Danish Government Institute of Seed Pathology, 78 Ryvamgs Alle, Hellerup, 2900 DK, Hellerup, Copenhagen, Denmark
Background and objectives
A large number of microtiter plates are needed for mass testing of seed lots for seed borne viruses. In case of poorly equipped laboratories. the possibility of re-use microtiter plates is not acceptable in ELISA for standard results in seed health testing. Petri-dish ELISA is an alternative technique based on the conventional ELISA. The indirect ELISA procedure was followed for comparison between the polystyrene solid phases of plastic petri-dish and microtiter plate (Nunce) . Three viruses belong to different groups of tobamovirus, comovirus and potyvirus were used to study the sensitivity of plastic petri-dish for detecting them in a very higher dilutions. A simple device used to divide the inner surfaces of plastic Petri-dish to many circles or squares by a hydrophobic substance e.g., normal wax or DAKO pen (used for immuno histochemical tests). The binding capacity of petri dish polystyrene for antigens was higher and proved sensitivity in detection of the above mentioned viruses. The modified method proved to be sensitive and acceptable to quick test at quarantine departments of the third world as well as for the researchers working in low facilities laboratories.
Results and conclusions
A comparison was made between polystyrene solid phase of petri dish and microtiter plate. Several dilutions from x100 up to x125,000 of sap prepared from leaf materials infected with 3 different viruses and from healthy leaves of corresponding plant species. The absorbance values (A 405nm) of all antigen-antibody reaction in petri dish and microtiter plate showed a complete agreement between positive and negative evaluations. Although, absorbance value of petri dish tended to be higher in the lower dilutions than microtiter plate. this differences were slightly increased at the higher dilution of TOMV and CPMV. Readings of BICPMV in petri dish was almost double compared with microtitre plate. Absorbance values (A405nm) for healthy sample were lower at all dilutions in petri-dish compared with microtitre plate. The present technique offer the following advantages over the conventional serological methods. (1) It requires a plastic petri-dish as a low cost solid phase. (2) consumption of a very little amounts of reagents. (3) it is more sensitive and accurate method compared with ELISA carried out on microtiter plate. (4) suitable for mass testing of seed lots for detecting seed borne viruses.
However, there are two disadvantage in this technique since the test is performed on petri-dish, it is difficult to measure the reaction automatically by ELISA reader but can be determined visually or measured after transfer to tubes by spectrophotometer. The second disadvantage is connected with the movement of the petri-dish from place while reagents inside it. The petri-dish must be handled carefully in a horizontal position without shaking.
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