Development of a PCR ‐Cas12a‐ LFD visual detection system for highly sensitive and specific detection of Ralstonia sp. , Phytophthora sp. , Alternaria sp. , and Pseudomonas sp. in tobacco

Abstract Ralstonia sp. , Phytophthora sp. , Alternaria sp. , and Pseudomonas sp. are the major pathogens responsible for tobacco diseases and severe threats to the sustainable development of the tobacco industry. Traditional detection methods, such as plate isolation and polymerase chain reaction (PCR), have improved in sensitivity and timeliness but remain limited by operational complexity, heavy reliance on high expertise requirements for result interpretation. This study focuses on the trans‐cleavage activity of the CRISPR‐Cas12a system and develops a series of novel detection methods for Ralstonia sp. , Phytophthora sp. , Alternaria sp. , and Pseudomonas sp. , respectively. By screening specific genomic targets, designing highly sensitive primers and CRISPR RNA (crRNA), and integrating PCR with lateral flow dipstick (LFD), the PCR‐Cas12a‐LFD detection system was established with a sensitivity of 100 pg μL −1 for Ralstonia sp. , 10 pg μL −1 for Phytophthora sp. , 5000 pg μL −1 for Alternaria sp. , and 0.1 pg μL −1 for Pseudomonas sp. , respectively, and excellent specificity without cross‐reactivity. Furthermore, the combination of this method with LFD enables visual interpretation of results. This study provides an efficient and visualized diagnostic tool for the early prevention and control of tobacco diseases during field cultivation while offering a theoretical reference for the molecular detection of other plant pathogens. © 2026 Society of Chemical Industry.