A Cocktail ELISA Semi-nested RT-PCR Assay to Detect Bean pod mottle virus in Soya bean Seeds

摘要

Bean pod mottle virus (BPMV) has been identified as an important pathogen for plant quarantine in China because large quantities of soya bean seeds (approximately 7x10(7) tons) are imported annually. To develop a practical detection programme for BPMV, a cocktail enzyme-linked immunosorbent assay (ELISA) nested RT-PCR using a combination of serological and molecular methods was designed for soya bean seeds. The single-vessel detection assay was performed in a 96-well ELISA plate, which served as a carrier for the subsequent nested RT-PCR assay. Assay specificity was demonstrated by the production of the expected 330- and 296-bp bands using the external and internal primers, respectively. This method was 10(4)-fold more sensitive than immunocapture-RT-PCR (IC-RT-PCR). In particular, it is important to note that this assay resulted in successful micro-extraction from soya bean seeds and combined the advantages of each individual technique. The cocktail ELISA nested RT-PCR is a specific, sensitive, rapid and economical procedure to rapidly identify and characterize BPMV and could be suitable for both primary-level platforms and laboratories.