An RPA-CRISPR/Cas12a-assisted method for nucleic acid detection of Haemonchus contortus in sheep

Abstract

Haemonchus contortus (H. contortus), a highly pathogenic and blood-feeding nematode, could cause haemonchosis，resulting in tens of billions of dollars in production losses and significantly impacting the development of sheep husbandry. Rapid and accurate detection methods were particularly important for the prevention and control of haemonchosis. In this study, we developed a one-pot effective detection method that integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology based on the conserved region of ITS2 of H. contortus, with readout through fluorescence signals visualized by lateral flow strips (LFS) and observable under UV or blue light. The detection procedure was successfully finished in within 1 h and demonstrated high specificity and sensitivity, with no cross-reactivity detected with nine other common ovine pathogens and a detection limit as low as 0.1 copies/μL for fluorescence and 100 copies/μL for LFS. Validation with 89 sheep fecal samples revealed a 46.07 % positivity rate, fully consistent with quantitative PCR results. In summary, the RPA-CRISPR/Cas12a method for H. contortus detection exhibited the advantages of high specificity, high sensitivity, and low device dependence, portable and visible results. The technique presented significant potential for large-scale clinical application and provided novel point-of-care testing for clinical use in remote rural and resource-constrained areas.