A multiplex PCR-Cas12a-lateral flow platform for pooled screening of four major tobacco pathogen groups.

Tobacco production is increasingly threatened by pathogens such as Ralstonia, Phytophthora, Alternaria, and Pseudomonas, which cause bacterial wilt, black shank, brown spot, and wildfire, respectively. Conventional diagnostics typically target single pathogens, and reliable methods for simultaneous multi-pathogen detection remain limited. Here, we developed a multiplex PCR-Cas12a-LFD platform for the pooled multi-target screening of four major tobacco pathogens. Multiplex PCR amplifies pathogen-specific 16S rRNA and ITS regions, while Cas12a-crRNA complexes specifically recognize the amplicons and trigger trans-cleavage of fluorophore-quencher or fluorophore-biotin probes, enabling fluorescence and lateral flow readouts. Reaction parameters were systematically optimized to reduce primer interference and achieve balanced amplification across targets. The individual target groups showed different mass-based analytical LODs, ranging from 5 fg/μL to 5 pg/μL under the tested conditions. For an equal-proportion mixed-template series, the universal reporter produced a pooled-signal LOD of 186 fg/μL and showed no detectable signal from the tested non-target DNA panel. LFD-based visual detection reached 0.5 pg/μL, while preliminary spiked-leaf tests at two extracted-DNA levels yielded apparent recovery rates of 93%-95%, indicating matrix compatibility under the tested conditions. This platform provides a laboratory-based pooled screening tool for preliminary sample triage, with target-specific identification requiring subsequent confirmatory analysis.