Babesia vogeli is a protozoan parasite causing canine babesiosis, a tick-borne disease prevalent in tropical and
subtropical regions. Its microscopic identification is challenging due to morphological similarity with other
Babesia spp., and serological assays often yield inaccurate results. To address this issue, we developed a rapid,
equipment-minimal diagnostic method combining recombinase polymerase amplification (RPA) with CRISPR/
Cas12a (RPA/CRISPR-cas12a) for B. vogeli-specific detection. The RPA assay enables DNA amplification for both
B. vogeli and Hepatozoon canis, while CRISPR/Cas12a using gRNA_Bab ensures specificity for B. vogeli, even in coinfections and other pathogens. This approach detects as few as 10? copies within two hours for both readout
platforms such as fluorescence and lateral flow dipstick (LFD). Forty canine blood samples were detected by
RPA/CRISPR-cas12a to examine its performance. Results showed high concordance with qPCR-high resolution
melting (HRM) (Cohen’s kappa: 0.93 for fluorescence, 0.81 for LFD), outperforming conventional PCR. The
clinical sensitivity and specificity of RPA/CRISPR-cas12a were 100 % and 96.8 %, respectively and the
concordance with qPCR-HRM was 97.5 %. RPA/CRISPR-cas12a for Babesia spp. detection provided a simple,
rapid, and accurate method, demonstrating promise for point-of-care diagnosis of canine babesiosis in resourcelimited settings. This method showed high potential as a practical diagnostic tool in veterinary clinics, with
accelerated surveillance to control outbreaks of Babesia-associated canine babesiosis.