Development of a reverse transcription loop-mediated isothermal amplification for detection of potato virus a in potato and in insect vector aphids

An isothermal-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of Potato virus A (PVA). The assay was optimized with respect to the selection of primer, optimization of isothermal temperature, and its incubation period. Visual detection of the LAMP reaction was also developed by adding SYBR gold nucleic acid staining dye, where the change in color from dark orange to fluorescent yellowish green indicates a positive reaction. The assay was examined for its specificity with other viruses-infecting potato and with the host genome and was found specific to PVA. The sensitivity of the assay was found equally sensitive to that of the RT-PCR assay. The optimized assay was validated for the detection of PVA from potato leaf samples collected randomly from fields. The assay was also successful in the detection of PVA from potato tubers. To determine the virus-carrying aphids, a Squash Print RT-LAMP (SP-RT-LAMP) assay was developed. The SP-RT-LAMP assay was successful in the detection of PVA in single aphids. It was further validated to detect the target virus in single aphid samples collected randomly from potato canopy. The assay revealed that 33 of 69 single aphids were carrying the virus. We concluded that the assay is simple and sensitive for the detection of PVA in potato leaf and tubers and also in insect vector aphids.