One-step reverse transcription loop-mediated isothermal amplification: a simple, sensitive and rapid assay for detection of potato virus X in potato leaves and tubers

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of potato virus X (PVX) targeting coat protein gene. The RT-LAMP assay was carried out using known positive (PVX), LAMP specific primers, amplification at 65 °C for 60 min, 80 °C for 10 min and visualized in agarose gel and viewed directly using SYBR gold staining dye. Change in color from dark orange to fluorescent yellowish green was observed only in the positive sample or by adding Hydroxynaphthol Blue dye where change in colour from violet to sky blue was observed. The assay was further optimized for the concentration of primers and components of reaction mixture, temperature, duration and finally the total volume of reaction mixture. Reduction in incubation time from 60 to 30 min and volume from 20 to 10 µl without compromising the sensitivity is a significant change. The assay was examined for its cross-reactivity with other viruses (PVY, PVM, PVA, PVS and PLRV) infecting potato where no cross-reaction was observed. The sensitivity of the LAMP assay was compared with RT-PCR, where the LAMP assay showed an increased sensitivity (10−5) i.e., 10 times more sensitive in comparison with RT-PCR (10−4) based assay. The optimized one-step and two-step LAMP assays successfully detected the target virus, PVX in potato tubers. Hence, the assay proved reliable, simple, specific, sensitive and rapid for detection of PVX in diagnostic laboratories. © 2019, Indian Phytopathological Society.