Rapid and multiple visual detection of Fasciola hepatica in feces via recombinase polymerase amplification integrated with CRISPR/Cas12a technology.

Fasciola hepatica is a foodborne zoonotic parasite causing significant economic losses and impacting human and livestock health in resource-limited regions. We developed a rapid, reliable, and sensitive detection method combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a, allowing visualization with the naked eye or a fluorescence reader. Multiple visual methods were used to analyze the assay results. Fluorescence signals were collected using a fluorescence reader or observed under UV or blue light. Lateral flow strips (LFS) were used for visual detection. Among seven primer pairs and three CRISPR RNA (crRNA) screened, F1/R1 and crRNA3 were optimal. The Cas12a reaction buffer was optimized with 50 mM Tris-HCl and 80 mM NaCl, with an RPA reaction time of 20 min. The assay showed high specificity and excellent sensitivity for F. hepatica, detecting 0.122 copies/μL with fluorescence and 8.6 copies/μL with LFS. Testing of 143 sheep and 43 human fecal samples showed 98.39 % consistency with qPCR and nested PCR, with prevalence rates of 52.45 % and 18.6 % in sheep and humans, respectively. Our assay offers substantial potential for point-of-care testing in resource-limited areas, addressing the need for rapid and accurate diagnosis of F. hepatica.