A rapid and specific strategy for detecting Orientobilharzia turkestanicum, a water-associated schistosome of veterinary and environmental concern.

Orientobilharzia turkestanicum (O. turkestanicum) is a water-associated schistosome parasite widely distributed in pastoral regions of Asia and Europe, where freshwater systems act as key interfaces linking livestock hosts, snail intermediate hosts, and environmental transmission pathways. Despite its strong environmental dependency, effective surveillance of O. turkestanicum in water-related settings remains limited by conventional diagnostic approaches that are time-consuming, equipment-dependent, and poorly suited for field-based monitoring. In this study, a rapid and sensitive detection assay based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (RPA-CRISPR/Cas12a) was developed for on-site identification of O. turkestanicum. The ITS1-5.8S region was selected as the molecular target to enable reliable species discrimination from closely related schistosomes, particularly Schistosoma japonicum (S. japonicum). The assay operates under isothermal conditions at 37 °C and allows result interpretation through visual fluorescence and lateral flow strip (LFS) readouts without the need for sophisticated instrumentation. The established RPA-CRISPR/Cas12a assay exhibited high analytical specificity, showing no cross-reactivity with a range of non-target parasites and bacterial species. Sensitivity evaluation using serially diluted standard plasmids demonstrated analytical limit of detection of 0.16 copies/μL by visual fluorescence and 160 copies/μL by LFS. Field applicability was validated using snail samples, cattle feces, liver, intestinal tissues and simulated infected water samples, yielding an overall concordance rate of 97.55 % compared with qPCR. Collectively, these results indicate that the proposed RPA-CRISPR/Cas12a assay provides a practical and field-deployable tool for livestock and water-associated transmission environment of O. turkestanicum and offers a useful framework for improving field surveillance and risk assessment of livestock schistosomiasis in endemic regions.