Visual and rapid detection of Fasciola spp. in fecal samples using a one-pot RPA-CRISPR/Cas12a assay

Fasciolosis caused by Fasciola spp. has substantial veterinary and public-health importance, while conventional fecal microscopy has limited sensitivity and PCR depends on laboratory equipment. This study aimed to develop a rapid, visual one-pot recombinase polymerase amplification (RPA)–CRISPR/Cas12a assay for detecting Fasciola DNA in fecal samples. RPA primers and CRISPR RNAs targeting the conserved internal transcribed spacer 1 region were screened and optimized. The final assay was completed at 37 °C within 30 min, and results were read using a fluorescence detector or visualized under blue light. The analytical limit of detection was 10 copies/µL, and DNA corresponding to 0.1 egg equivalents was detectable. No cross-reactivity was observed with six non-target parasite species. Among 54 field fecal samples (42 yak and 12 Tibetan sheep samples) from Xizang, three were positive, with complete qualitative agreement between the one-pot assay and conventional PCR in this limited sample set. In a preliminary mixed-DNA interference evaluation using 24 artificially spiked fecal samples, all 23 samples containing Fasciola DNA, including samples containing DNA from both Fasciola species or Fasciola DNA combined with selected non-target parasite DNA, were positive, whereas the non-target-only control was negative. The one-pot RPA–CRISPR/Cas12a assay provides rapid and analytically sensitive detection of Fasciola DNA, with no cross-reactivity observed within the tested parasite panel. The method shows potential for surveillance and resource-limited testing, but broader multi-region validation and simplified sample preparation are required before routine field or point-of-care deployment.