Field-deployable multiplex RAA-CRISPR/Cas12a platform rapidly and simultaneously detects seven Eimeria species in chickens.

Chicken coccidiosis, caused by infection with one or more of the seven Eimeria spp., is a major challenge in global poultry production. Rapid and accurate identification at the species level is critical for guiding targeted treatment strategies, minimizing antibiotic misuse, and mitigating disease transmission. In this study, we developed a point-of-care testing (POCT) platform, E-MRC12a (Eimeria-Multiplex RAA-CRISPR/Cas12a), which integrates multiplex recombinase-aided amplification (RAA) with CRISPR/Cas12a technology for the simultaneous detection of all seven Eimeria species in chicken fecal samples. Key assay parameters were optimized to balance detection performance and operational cost. The system was comprehensively evaluated for its sensitivity, specificity, reproducibility, and field applicability. E-MRC12a enables visual, one-pot detection of as few as 1 oocyst/μL. The process from sample loading to result interpretation required 1 h, while the total time from initial sample processing to final result readout was approximately 2 h. The assay exhibited high specificity with no cross-reactivity among Eimeria species, and demonstrated 100% concordance with conventional diagnostic methods in clinical validation. This rapid, field-deployable platform provides a species-specific coccidiosis diagnostic solution, supporting epidemiological surveillance and multivalent anticoccidial vaccine development.