A Rapid Visual Detection Method for Fasciola hepatica Based on RAA-CRISPR/Cas12b

Area of Science:

• Parasitology
• Molecular Diagnostics
• Biotechnology

Background:

• Fascioliasis is a significant zoonotic disease impacting public health and livestock.
• Existing diagnostic methods require specialized equipment and expertise, limiting their use in resource-limited areas.

Purpose of the Study:

• To develop a rapid, visual, and field-deployable diagnostic assay for Fasciola hepatica.
• To overcome the limitations of conventional diagnostic techniques for on-site surveillance.

Main Methods:

• Development of a recombinase-aided amplification (RAA) assay targeting Fasciola hepatica mitochondrial DNA.
• Integration of CRISPR/Cas12b technology for enhanced specificity and detection.
• Optimization of primer and sgRNA sequences, and development of three visual detection modalities (real-time fluorescence, UV lamp, test strip).

Main Results:

• The assay achieved a rapid turnaround time of 45 minutes.
• Demonstrated high specificity for Fasciola hepatica with a real-time fluorescence sensitivity of 2.6 copies/μL.
• Showed high concordance with established PCR and qPCR methods, indicating reliable performance.

Conclusions:

• The developed RAA-CRISPR/Cas12b assay offers a sensitive, specific, and visually interpretable method for Fascioliasis detection.
• This assay overcomes the constraints of traditional diagnostics, providing an efficient tool for grassroots and pastoral regions.
• Enables effective field-deployable surveillance and control of Fascioliasis.