Development of rapid and simple detection of bean common mosaic virus (BCMV) in mung beans (Vigna radiata) using reverse transcription-loop mediated isothermal amplification (RT-LAMP).

Abstract

Bean common mosaic virus (BCMV) is one of the most serious and devastating Potyvirus of leguminous crops. In mung bean (Vigna radiata), BCMV is an emerging virus causing enormous losses to the crop, thereby reducing the production and profitability of the crop. Being seed borne and aphid transmitted virus, it important to reduce the spread and prevent its transfer to new geographical locations using rapid, specific and sensitive detection techniques. In this study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was devised to rapidly and specifically detect BCMV. Three pairs of specific primers were designed targeting the BCMV genome. To determine the ideal temperature, reactions were carried out across a temperature range of 45 °C to 70 °C, with intervals of 5 °C. The optimal temperature for the assay was determined to be 60 °C with a 30-min incubation period. Comparison between the RT-LAMP and conventional reverse transcription polymerase chain reaction (RT-PCR) revealed that former can detect the BCMV upto 10^- 9 and was one hundred times more sensitive than later. It was also determined that RT-LAMP was specific only in detecting BCMV, with no cross-reactivity with other closely related non-target viruses [potato virus Y (PVY), bean common mosaic necrosis virus (BCMNV), clover yellow vein virus (ClYVV) and soybean mosaic virus (SMV)]. After incubating the reactions at constant temperature of (60 °C/30 min), a characteristic ladder like banding pattern was observed on agarose gel for positive samples. Colorimetric tests (SYBR Green I) were also performed to reduce the requirement of laboratory equipment for visualizing RT-LAMP results. The results developed by SYBR Green I were comparable to that of agarose gel and can be visualized with naked eye. The developed RT-LAMP assay enables rapid detection of BCMV at 60 °C within a time period of 30-min.