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Updated: Oct 25, 2026
Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a
Published on: December 23, 2022
RNase H-Assisted, Split-crRNA-Based CRISPR/Cas12a Lateral Flow Assay for Amplification-Free, Multi-Site Detection of
Yoo-Hong Min1, Deok-Gyu Lee1,2, Jin-Hee Yoo1,2
1Critical Diseases Diagnostics Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon34141, Republic of Korea.
Abstract:
With the growing demand for on-site testing of foodborne pathogens, rapid and sensitive detection methods are urgently needed. Here, we developed a split-crRNA-based CRISPR/Cas12a assay integrated with a lateral flow assay (sCR-LFA) for rapid, sensitive detection of bacterial 16S rRNA within 1 h, without pre-amplification. To detect highly structured 16S rRNA, we used RNA fragmentation via DNA-helper-mediated RNase H pretreatment. When short RNA targets interact with corresponding RNA blockers, unblocked RNA spacers hybridize with a DNA activator to form spacer/activator duplexes, which activate split-crRNA/Cas12a complexes. The sCR-LFA achieved a detection limit of 104 CFU/mL for single-site targeting. Using an enhanced sCR-LFA that integrates a multi-site targeting strategy, we achieved a lower detection limit of 102 CFU/mL. Furthermore, the enhanced sCR-LFA was successfully validated in simulated milk and mushroom samples spiked with bacteria. Collectively, this work presents a promising approach for effective food safety monitoring and prevention of infectious diseases.
