Loop-mediated isothermal amplification assay for quicker detection of tomato leaf curl Joydebpur virus infection in chilli

Abstract

Chilli leaf curl disease caused by whitefly transmitted begomoviruses is an important constraint to chilli (Capsicum anuum L.) cultivation in India. Tomato leaf curl Joydebpur virus (ToLCJoV) was characterized and identified as incitant of leaf curl disease through rolling circle amplification (RCA) and PCR assay from the symptomatic samples collected from Uttar Pradesh, India. Although PCR assay provides the gold standard in diagnostics, this method consumes more time and requires convenient portable instruments. Therefore, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of ToLCJoV by targeting the AC1 and AC2 region. Detection has been achieved through a laddered pattern of amplification in agarose gel electrophoresis. The assay has detected ToLCJoV in a total DNA concentration of 1 × 10−1 ng indicating 200-fold higher sensitivity than that of the PCR. Further, the replacement of total DNA with leaf extracts using the grinding buffer and GES buffer coupled with LAMP assay also detected the presence of ToLCJoV in the infected chilli samples. With this assay, ToLCJoV can be detected in less than 2 h without DNA extraction. Besides, this assay will be highly useful in discriminating the leaf curl disease etiology by ToLCJoV from other begomoviruses and insects (thrips and mites). To the best of our knowledge, this is the first report of a LAMP assay for the detection of ToLCJoV.