Babesia bovis is a known causative agent of bovine babesiosis and is widely distributed across China. Rapid detection and accurate identification of B. bovis is essential for follow-up management and epidemiological investigations. In this study, a cross-priming amplification combined with vertical flow (CPA-VF) assay was developed. The detection limit of the CPA-VF assay targeting the 18S rRNA gene was 320 fg per reaction at 61 °C for 60 min. No cross-reactions were observed with other piroplasms infective to cattle. Furthermore, 36 blood samples from experimentally-infected animals were accurately assessed using the CPA-VF assay. The performance of the CPA-VF assay was compared with the results of conventional PCR for 219 blood samples from the field. Our results demonstrate that the CPA-VF assay is a practical and effective diagnostic tool for bovine babesiosis caused by B. bovis infection.
