Research regarding the improvement of methods used for potato viruses Y, A, X, S and PLRV identification

Abstract

Obtaining health and safety food impose the improvement of identification’s techniques of pathogen agents. The goal of this research was to examine and evaluate the effects of several modifications of ELISA (enzyme linked immunosorbent assay) technique on the detection of potato viruses Y, A, X, S and potato leafroll virus (PLRV). These modifications consisted on: the use of sap extracted directly from the tubers, modification of the incubation modality of conjugate (IgG-AP), the use of several additives in extraction and conjugate buffers, replacement of grinding buffer with McIlvain buffer. The results show a better identification of PLRV in sprouting tubers using the co-incubation sample and IgG-AP conjugate. Compared with the classical method, the test safety and sensitivity increased. Using sap from sprouting tubers (dilution 1/10) the average values of OD at 405 nm was 2.5 times higher. The detection of potato viruses Y and A by enzyme– linked immunosorbent assay (ELISA) can be improved using extraction buffers with new composition. Using McIlvain’s phosphate-citric acid buffer (0.18M; pH 7), the absorbance values (A405nm) increased significantly for PVY and PVA detection comparing with the classic extraction buffer. Sodium diethyldithiocarbamate (0.01M) in phosphate-buffered saline plus Tween 20 (PBS-T) used instead of the polyvinylpyrrolidone increased the sensitivity of potato virus Y but this additives decrease the absorbance values in case of PLRV identification. The same decrease was observed when we used sodium thioglicolat (0.01M) and sodium diethyldithiocarbamate (0.01M) in PBST. Presence of proteins in conjugate buffer improve safety of viruses identification. Food gelatin was more efficiently like the bovine serum albumine (BSA) for PLRV identification. The use of new equipement, the use of McIlvain’s buffer and gelatin food could save time and costs in routine diagnostic of viruses wich affect potato plants.