Development of a new PCR assay and a recombinase-aided amplification based isothermal amplification coupled with lateral flow dipstick assay for potato late blight detection

Abstract

Late blight caused by Phytophthora infestans is the most devastating potato disease, yet leaf infections are not reliably detected by current assays during the incubation phase of the pathogen and asymptomatic plants. In this study, the genome of P. infestans was queried against the NCBI nucleic acid database and a 1050 bp species-specific DNA fragment was identified. Using this fragment, polymerase chain reaction (PCR) and recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assays were developed for the detection of potato late blight pathogen. Both assays effectively distinguished P. infestans from related species (e.g., P. capsici). The detection limits of the three PCR primer pairs under optimized conditions (59.3 °C, 35 PCR cycles) were 1.31 × 10−1 fg, 1.18 × 10−2 fg, and 1.24 × 10−1 fg, corresponding to 27.31 copies, 2.49 copies, and 25.51 copies, respectively. While the detection limit of the RAA-LFD assay at 37 °C for 25 min was 5 × 10−4 fg (0.10 copies), which was better than all reported molecular detection assays for potato late blight. Both PCR (59.3 °C, 35 PCR cycles) and RAA-LFD (37 °C for 10 min) positively detected all symptomatic leaves. When extending the amplification time to 20 min, the RAA-LFD assay detected most of the leaves with incubation phase of the disease. The molecular detection assays developed in this study were species-specific and are expected to be used in detecting potato late blight in the incubation phase in leaves and asymptomatic plants, which will aid in accurate monitoring and early warning of potato late blight.