A cost-effective and rapid TE-based DNA extraction for near point-of-care detection of Babesia spp. by RPA/CRISPR-Cas12a.

BACKGROUND: Babesia spp. infects a wide range of mammals, including domestic animals, wildlife, and humans, highlighting the need for rapid diagnostics. This study developed a near point-of-care method combining rapid DNA extraction, recombinase polymerase amplification (RPA), and CRISPR-Cas12a for Babesia detection. METHODS AND RESULTS: The workflow includes three steps: DNA extraction, RPA amplification, and CRISPR-Cas12a signal detection. Five blood DNA extraction methods were evaluated: DNeasy kit, boiling, Tris-EDTA (TE) buffer, methanol-based, and Chelex®100. The commercial kit, boiling method, and TE buffer extraction yielded positive results using RPA/CRISPR-Cas12a based on the cytochrome c oxidase subunit I (COI) gene. The TE buffer extraction and boiling methods were further optimized by RPA/CRISPR-Cas12a assay using Babesia-specific primers based on the 18S rRNA gene. All these extraction methods produced results comparable to the commercial kit. The TE buffer extraction was selected due to its simplicity and speed. The RPA/CRISPR-Cas12a assay specifically detected Babesia spp., including B. vogeli, B. gibsoni, and B. ovata. The limit of detection using TE-extracted DNA was 104 copies per reaction, lower than that achieved with the commercial kit (103 copies per reaction). Evaluation of rapid DNA extraction for Babesia spp. detection by the RPA/CRISPR-Cas12a assay using seven bovine and eight canine blood samples showed complete concordance between the TE buffer extraction and the commercial kit, with six positive samples detected. CONCLUSIONS: The TE buffer extraction offered a low-cost, simple, and equipment-minimal approach suitable for combination with RPA/CRISPR-Cas12a diagnostics, enabling rapid detection of Babesia spp. in resource-limited or field settings.