Rapid visual detection of Moniezia spp. in sheep feces via Recombinase Polymerase Amplification-Lateral Flow Dipstick (RPA-LFD) assay

Monieziasis is a prevalent issue in small ruminant husbandry, primarily caused by Moniezia expansa and M. benedeni in China. There is a critical need for highly sensitive methods for disease surveillance and prevention. In this study, we developed a visual assay combining recombinase polymerase amplification (RPA) with a lateral flow strip (RPA-LFD) for the rapid detection of Moniezia spp. in sheep fecal samples. Seven primer/probe sets were designed based on a specific fragment of the M. expansa β-tubulin gene (MTUB). The RPA-LFD assay performed optimally under reaction conditions of 39 ℃ for 15 min, with a primer-to-probe ratio of 4:0.6. The optimized method demonstrated high specificity for Moniezia spp., with no cross-reactivity with genomic DNA from other common gastrointestinal parasites in ruminant livestock. The detection limit was 10 copies/μL of plasmid DNA or 10 pg/μL of M. expansa genomic DNA per reaction. When compared to PCR using clinical and sheep-simulated samples, the RPA-LFD assay exhibited equivalent detection capability, achieving 95.7 % consistency (K value = 0.939, p < 0.001). These results suggest that the RPA-LFD method is a reliable and portable diagnostic tool for routine screening, monitoring, and rapid confirmation of monieziasis in sheep flocks, particularly in endemic areas and remote regions.