Fascioliasis, a globally prevalent zoonosis, severely threatens public health and livestock security. Current diagnostic approaches, hindered by the need for sophisticated instrumentation and specialized expertise, are inadequate for on-site surveillance in resource-constrained settings. This study developed a rapid, visual detection assay for Fasciola hepatica via recombinase-aided amplification (RAA) integrated with CRISPR/Cas12b, addressing critical equipment and operational constraints. Targeting a specific mitochondrial DNA fragment of F. hepatica, recombinant plasmid standards were constructed, RAA primers and sgRNA optimized, and three detection modalities (real-time fluorescence, UV lamp, test strip) integrated. Clinical validation against PCR demonstrated 45 min turnaround time, F. hepatica-specific positivity, and real-time fluorescence sensitivity of 2.6 copies/μL. Results showed high concordance with PCR and qPCR, with substantially reduced assay duration and streamlined workflow. This highly sensitive, specific, multi-visualized method overcomes limitations of conventional techniques, offering an efficient, field-deployable tool for fascioliasis surveillance and control in grassroots and pastoral regions.

This review systematically summarizes recent advances in CRISPR-Cas12b-based pathogen nucleic acid detection. Starting with an overview of conventional nucleic acid detection methods and core CRISPR-Cas mechanisms, we highlight the unique properties that distinguish Cas12b from other Cas effectors. We elaborate on the working principles of the CRISPR-Cas12b system and present comparative structural and functional analyses with other Cas variants to underscore its distinctive molecular architecture and enzymatic properties. A comprehensive evaluation of current applications demonstrates the efficacy of CRISPR-Cas12b-based diagnostics across diverse pathogens, including viruses, bacteria, and parasites, with a specific focus on its integration with isothermal amplification techniques. We further examine the translational potential of CRISPR-based diagnostics in clinical settings, while critically analyzing persistent technical challenges including off-target effects, signal amplification limitations, and sample preparation requirements. Targeted strategic recommendations are proposed to optimize detection sensitivity, develop multiplexed detection platforms, and implement point-of-care testing configurations. This review aims to systematically correlate the unique characteristics of Cas12b with its broad diagnostic applications, thereby addressing key research gaps in its progression toward clinical validation and field deployment. It also provides a targeted framework to accelerate the translation of this technology from laboratory platforms to practical diagnostic solutions.