Development and application of fluorescent loop mediated isothermal amplification technique to detect Phytophthora infestans from potato tubers targeting ITS-1 region.

Abstract

The goal of this study was to develop a fluorescent based loop-mediated isothermal amplification (LAMP) assay for a simple, sensitive and visual detection of P. infestans from tubers targeting a novel internal transcribed spacer 1 (ITS-1) region of ribosomal DNA. The ITS-1 LAMP primers were designed using the Primer Explorer V4 software. The optimization of LAMP reaction conditions and reagents concentrations were carried out with time, temperature, MgSO4, dNTPs and WarmStart Bst DNA polymerase. The amplified products were analysed using SYBR Green I dye and by agarose gel electrophoresis. We optimized reaction conditions included reagent mix, incubated at 65°C for 60min. The target specificity of primers was assessed with PCR, restriction digestion and sequence analysis. The developed LAMP assay was evaluated for its analytical specificity, sensitivity and validation in field tuber samples. The analytical specificity of LAMP primers indicates positive reaction with P. infestans and closely related species except P. erythosepctica. We were able to detect down to 1pg/µl of DNA using the newly developed LAMP primers whereas the minimal amount detectable for conventional PCR was 0.1ng/µl. Further, the samples with positive reaction developed a characteristic fluorescent green color. The detection of LAMP assay for inoculum of P. infestans was determined in the artificially inoculated leaves and tubers. In 98 field tuber samples, 54 (55.10%) were confirmed as positive by LAMP while 39 (39.79%) positive by PCR. The LAMP assay developed in this study has a potential to be a beneficial tool in early detection of P. infestans in low cost laboratory. Because the LAMP assay performed well in aspects of sensitivity, repeatability, target specificity, reliability, and visibility, it is suitable for detection of P. infestans in infected potato tubers.