Development of an ERA-CRISPR/Cas12a-based visual diagnostic assay for bovine babesiosis

Bovine babesiosis, caused by Babesia bovis, is a tick-borne hemoparasitic disease causing substantial economic losses to cattle industries worldwide. Current diagnostic methods, particularly microscopy, lack sensitivity for subclinical infections, while molecular assays require sophisticated equipment unsuitable for field use. To address this gap, we developed a novel diagnostic assay integrating enzymatic recombinase amplification (ERA) with CRISPR-Cas12a-mediated trans-cleavage, targeting the B. bovis spherical body protein 2 (SBP2) gene. The assay enables visual readout via UV/blue light fluorescence and lateral flow strips. Following systematic optimization of primers, crRNA, reaction temperature, and reporter concentration, the ERA-CRISPR/Cas12a system achieved complete amplification within 20 min at 37 °C and demonstrated a detection limit of 10 copies/μL with high specificity for B. bovis. comparable diagnostic performance with conventional PCR within the tested sample panel. The fluorescence readout achieved a detection limit of 10¹ copies/μL, while the lateral-flow strip format exhibited a detection limit of 104 copies/μL. The entire procedure, from DNA extraction (approximately 30 min) to final result readout (ERA: 20 min; CRISPR-Cas12a: 20 min), can be completed within approximately 70 min. This detection method supports two types of visual detection: portable blue light and test strips, and is suitable for on-site environments with limited resources. This work provides a practical diagnostic tool for rapid, accurate detection of bovine babesiosis, with potential applicability to other veterinary pathogens.