Detection of viruses in seeds of some vegetables by reverse transcriptase polymerase chain reaction (RT-PCR)

Abstract

The present study was carried out to detect the presence of Alfalfa mosaic alfamovirus (AMV), cucumber mosaic cucumovirus (CMV), lettuce mosaic potyvirus (LMV), cucumber green mottle mosaic virus (CGMMV), tomato bushy stunt tombusvirus (TBSV), tobacco mosaic tobamovirus (TMV), tomato black ring nepovirus (TBRV) and tomato mosaic tobamovirus (ToMV) in seeds of pepper, tomato, cucumber and lettuce which are essential for human nutrition and seed production in Turkey. 50 seed samples for each vegetable obtained from various foundations and farmers were tested by reverse transcriptase polymerase chain reaction (RT-PCR) and double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) was conducted to compare the results. Besides, two total nucleic acid (TNA) extraction methods and a commercial RNA purification kit was compared to get high quality RNA for optimized RT-PCR. Purified total RNA extracts were used as template for cDNA synthesis, PCR products were analyzed by gel electrophoresis, and visualized by ethidium bromide staining. As a result of all, the optimum TNA extraction method was silica capture which is practical and easy to use for getting viral agents from tested vegetable seeds. RT-PCR was found rapid and sensitive in detecting viruses in seeds of vegetables when compared to DAS-ELISA. This study shows that RT-PCR can be successfully applied in certification programs and quarantine tests of vegetable seeds.

Keywords: Seed, reverse transcriptase polymerase chain reaction (RT-PCR), total nucleic acid (TNA) extraction, optimization

African Journal of Biotechnology Vol. 12(25), pp. 3891-3897