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.
