开发一种一锅式LAMP-CRISPR/Cas12b平台，用于快速现场检测破伤风梭菌

Abstract

Clostridium perfringens (C. perfringens) is a major causative agent of foodborne diseases, yet the practical deployment of loop-mediated isothermal amplification (LAMP) for its rapid on-site detection remains constrained by non-specific amplification and aerosol contamination. In this study, we established and systematically optimized a one-pot LAMP–CRISPR/Cas12b platform that integrated target amplification and sequence-specific verification within a single one-pot reaction, enabling accurate molecular detection without sophisticated instrumentation. Targeting the cpa virulence gene, the assay completed detection within 30 min and achieved a limit of detection of 2.74 × 10 1 copies/μL, as determined by Probit regression analysis with 20 replicates per concentration. No cross-reactivity was observed with other common foodborne pathogens, demonstrating high analytical specificity. Validation using 15 bacterial culture samples and 60 food samples showed complete concordance with quantitative PCR (qPCR) and conventional LAMP assays. Importantly, CRISPR/Cas12b-mediated secondary sequence verification effectively suppressed false-positive signals arising from non-specific LAMP amplification, while the closed-tube format significantly reduced the risk of aerosol contamination and streamlined operation. By combining rapid isothermal amplification with highly specific CRISPR-based molecular recognition in a closed-tube workflow, this platform provides a sensitive, robust, and field-deployable strategy for point-of-care detection of C. perfringens . The proposed approach also provides a reference strategy for adapting one-pot CRISPR-based isothermal assays to other foodborne pathogens in food safety monitoring.