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The influence of samples incubation on detection of PLRV and the effect of some extraction buffer’s additives on the detection of potato viruses Y, A, X and by ELISA technique

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Author(s): Carmen Liliana BADARAU | Nicolae COJOCARU | Sorin Nicolae RUSU | Maria IANOSI | Krisztina PETRUSCA

Journal: Analele Universitatii din Oradea, Fascicula Biologie
ISSN 1224-5119

Volume: TOM XVI;
Issue: 2;
Start page: 15;
Date: 2009;
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Keywords: potato virus | co-incubation | extraction buffer | ELISA

ABSTRACT
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 by this technique. These modifications consisted on: modification of the incubation modality of conjugate (IgG-AP), the use of several additives in extraction buffer, replacement of grinding buffer with McIlvain buffer. The results show a better identification of PLRV in leaves and sprouting tubers using the co-incubation sample and IgG-AP conjugate. Compared with the classical method, the test safety and sensitivity increased. Testing leaves, the average values of OD at 405 nm was 5-6,6 times higher than those obtained by standard DAS ELISA method and using sap from sprouting tubers (dilution 1/10) this average was 1,5 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 PBS-T. The co-incubation sample and conjugate and the use of McIlvain’s buffer could save time and costs of potato viruses diagnostic tests.
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