Development of a visual detection method for Potato virus S by reverse transcription loop-mediated isothermal amplification.

Abstract

A reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay was developed to detect the Potato virus S (PVS) in potato. Two sets of six novel primers that recognize the coat protein gene sequence of the PVS were designed and RT-LAMP assay was optimized for the parameters such as different concentrations of primers, MgSO4, betaine, dNTPs, Bst DNA polymerase, temperature and duration. The RT-LAMP was carried out under isothermal conditions without the thermal cycler using PVS infected leaf and tuber samples, LAMP specific primers with amplification at 65°C for 60min, and 80°C for 5min. The results were assessed by gel electrophoresis and visual observation of colour change using SYBR Green I dye. The detection limit of the developed RT-LAMP assay was determined and compared with a conventional reverse transcription-polymerase chain reaction (RT-PCR). RT-LAMP was found 100 times more sensitive than RT-PCR. The optimized RT-LAMP assay is robust, reliable, sensitive and convenient for the detection of the PVS in infected potato tubers including asymptomatic plants. No cross-reactions were observed with healthy plants and other potato viruses. The assay is economical and can be employed in large scale testing of potato plants against PVS under healthy seed potato production programme.