Plant viruses are among the unique pathogens in terms of transmission, dissemination, and crop loss. Viruses transmit easily in nature via insect vectors, pollens and planting materials and no efficient control measures have been developed so far to combat these agents. Indeed, prevention of plants/planting materials from infection by viruses and virus-like agents is the only efficient method to safeguard the forthcoming agronomy and horticulture industries. However, this is crucial for plants that propagate vegetatively including potatoes. In fact, early-stage detection of viruses by sensitive and applicable methods to test thousands of samples in a certain time of a certification program is necessary to manage virus detection.

Several detection methods based on viral proteins, nucleic acids and their biology have been developed for virus detection in plants. Due to several differences in their sensitivity, cost and time to deliver test results, only a few techniques are used in applied plant certification. The Enzyme-linked immunosorbent assay (ELISA) is one of the methods that uses normally for virus detection in certification programs worldwide. However, ELISA assays demand commercially virus specific antibodies.

Based on the Iranian National Standards for Planting Materials and according to their class of certification, more than 120 thousand of seed potato tubers and or leaves are annually tested for six viruses and other pathogens that transmit on or in tubers. Viruses include Potato virus Potyvirus (PVY), Potato virus S Carlavirus (PVS), Potato virus M Carlavirus (PVM), Potato virus X Potexvirus (PVX), Potato virus A Potyvirus (PVA), and Potato leaf roll Polerovirus (PLRV). Testing for these viruses are normally done by DAS-ELISA using commercially available antibodies. However, in some cases, the results have to be rechecked by RT-PCR using virus-specific primer pairs.

Access to virus-specific antibodies have been the biggest challenge for potato seed certification in regard to their cost, delivery and accessibility in certain time. To manage this challenge, we tried to develop native antibodies for all six viruses using their recombinant coat protein (CP) expressed in bacterial systems. To this aim, isolates of PVY, PVA, PVS, PVX, PVM and PLRV were collected from all areas of Iran that produce seed and or table potatoes. The CP genes of several isolates from each virus, based on their origin and symptom manifestation, were amplified by RT-PCR and the PCR products were sequenced. The CP genes of dominant isolate (s) were amplified by specific primer pairs carrying restriction endonucleases and subsequently cloned in cloning vector. The cloned CP fragments were ligated into specific expression vectors and inserted into the appropriate Escherichia coli strains capable of foreign protein expression. The recombinant CP proteins were injected individually in rabbits and viral-specific antibodies were extracted and purified.

The specificity of the IgGs were tested by PAGE, Western-blotting and several other serological assays. Up to now, the efficiency of prepared antibodies were tested successfully on more than 200 thousands potato leaf and tuber samples and compared with commercial antibodies. At the moment we use these native antibodies for routine certification of all potato samples in serological assays.