One-step reverse transcription loop-mediated isothermal amplification assay for rapid detection of melon yellow spot virus

Abstract

A one-step, accelerated reverse transcription loop-mediated isothermal amplification (RT-LAMP) procedure was developed for the detection of Melon yellow spot virus (MYSV). The four primers required for accelerated RT-LAMP were designed to anneal onto a conserved region in MYSV N gene. Reaction temperature, optimal Mg2+ concentration, and the ratio of outer/inner primers affected reaction efficiency. MYSV RT-LAMP optimal reaction parameters were obtained in this study. The RT-LAMP was specific and sensitive in detecting MYSV in 60 minutes, with a detection limit of 0.5 × 10−4 μg total RNA per reaction, and a ladder-like pattern observed after agarose gel electrophoresis. RT-LAMP was 100-fold more sensitive than the conventional one-step reverse transcription polymerase chain reaction (RT-PCR) using serial dilutions of total RNA. RT-LAMP was a specific, sensitive, and low cost diagnostic tool that should be of value in more accurate determination of the distribution and host range of MYSV.