Babesiosis, a globally significant tick-borne disease, poses substantial threats to livestock production and public health. Reported cases of human babesiosis in the United States increased from 1742 in 2014 to 3586 in 2023. In livestock, cattle babesiosis causes mortality, reduced meat and milk production, reproductive losses, and substantial control costs, with annual economic losses estimated at hundreds of millions of US dollars in several endemic countries. Rapid and sensitive detection methods are essential for early warning, surveillance, and control of this disease. In this study, we developed a closed-tube, one-pot assay for genus-level detection of Babesia spp. associated with cattle and sheep, based on recombinase polymerase amplification (RPA) coupled with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a. This format effectively minimizes cross-contamination risks associated with repeated tube opening in conventional assays. A three-channel signal readout system, including blue-light fluorescence visualization, ultraviolet (UV) fluorescence visualization, and lateral flow strip (LFS) readout, was integrated to enable flexible endpoint detection under different laboratory and field conditions. The assay targets a conserved region of the Babesia 18S rRNA gene and enables genus-level detection of Babesia spp. within 40 min at 37°C. The established RPA-CRISPR/Cas12a platform exhibited high analytical sensitivity, with a limit of detection of 5 copies/μL for recombinant plasmid templates, high analytical specificity against the tested nontarget pathogens, and low equipment dependency. The detection limit of the LFS format reached 50 copies/μL. Field validation using 71 pooled tick samples and 53 clinical blood samples collected from cattle and sheep yielded positive rates of 15.49% and 9.43%, respectively, with 100% concordance between this assay and conventional polymerase chain reaction (PCR) for both specimen types. In conclusion, this one-pot RPA-CRISPR/Cas12a detection platform provides a rapid, sensitive, and field-applicable molecular screening tool for genus-level detection of Babesia spp. This assay may support early warning and preliminary field monitoring of babesiosis, particularly in resource-limited settings. However, species-level confirmation should be performed by sequencing or other species-specific methods when epidemiological tracing or precise species identification is required.
