Sensitive and rapid detection of peach latent mosaic viroid by the reverse transcription loop-mediated isothermal amplification

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of peach latent mosaic viroid (PLMVd) was developed. Four primer sets (OLD, OLD1, NEW, and Fukuta's) were designed originally. Based on initial experiments the set OLD1 was selected for further evaluation. Simple and accelerated RT-LAMP was preformed using degenerate and no degenerate forward-loop (F-loop) and backward-loop (B-Loop) primers. Degenerate primers were selected, and after determination of their best concentration (0.8 μM), the reaction was preformed at different temperatures (60–67.5 °C) using three different betaine concentrations (0.8 M, 0.4 M, and 0.2 M). Optimal conditions were found to be 62.5 °C and 0.8 M betaine. Under these conditions, using tRNA as template, PLMVd was detected within only 32 min, compared to 180 min of RT-PCR, using the Real Time Turbimeter (LA200, Teramecs) which measures the turbidity caused by the production of insoluble magnesium pyrophosphate. In addition, RT-LAMP was more sensitive than RT-PCR. PLMVd was detected in peach, plum, apricot, pear, wild pear and quince samples.