A Handy and Visual Detection Method for Potato Late Blight Utilizing Multi-Copy Specific Sequences and RPA-LFD...

Potato late blight, caused by Phytophthora infestans, is one of the most destructive diseases affecting global potato production. Rapid and accurate detection of the pathogen is essential for effective disease prevention and control. In this study, a novel multi-copy and specific DNA sequence, designated SSPI1 (Specific Sequence of Phytophthora infestans 1), was identified through bioinformatic analysis. Using seven closely related Phytophthora species and two additional common potato pathogens as controls, PCR assays demonstrated that SSPI1 offers higher sensitivity than PiYPT1 and improved specificity over PiITS. Based on this novel multi-copy sequence, we developed a rapid, user-friendly, and highly sensitive detection method for potato late blight by combining recombinase polymerase amplification (RPA) with lateral flow dipstick (LFD) technology. The entire diagnostic process-including crude DNA extraction, RPA amplification, and visual detection-can be completed within 35 min without the need for specialized equipment. Optimization results indicated that among nine candidate RPA primer pairs, RPA-F1/RPA-R2 exhibited the highest amplification efficiency. The optimal RPA reaction temperature range was determined to be 27-42°C, with a detection limit of 11 copies/μl for SSPI1. This method could detect as little as 100 pg of genomic DNA, identify samples collected as early as 24 h post-inoculation, and even detect infections in asymptomatic leaves under field conditions, demonstrating superior sensitivity compared to the PiYPT1-targeted RPA-LFD assay. Overall, this study presents a highly sensitive, specific, practical, and efficient tool for the early and rapid detection of potato late blight.