Rapid Detection of Chicken Infectious Anemia Virus Using a One-Tube RPA-CRISPR/Cas12a System

Chicken infectious anemia virus (CIAV) is a major immunosuppressive pathogen that causes significant economic losses to the global poultry industry. Conventional detection methods for CIAV are limited by poor timeliness, high equipment requirements, and insufficient sensitivity. To address these challenges, this study developed a novel one-tube integrated RPA-CRISPR/Cas12a assay targeting the highly conserved VP3 gene of CIAV for rapid and accurate detection. The performance of the assay was comprehensively evaluated in terms of sensitivity, specificity, and repeatability. Its clinical utility was assessed by testing 80 clinical suspected samples, with quantitative real-time PCR (qPCR) serving as the reference method. The results showed that the limit of detection (LoD) of the developed method was 10 copies/reaction, comparable to that of qPCR. No cross-reactivity with common avian pathogens was observed. The intra- and inter-assay coefficients of variation (CV%) for the time to threshold (Tt) were both below 10%. In clinical sample detection, the assay achieved a total coincidence rate of 97.5%, with a sensitivity of 100% and specificity of 96% relative to qPCR. In conclusion, the RPA-CRISPR/Cas12a assay developed in this study offers rapid detection, high sensitivity and specificity, operational simplicity, low equipment dependency, and excellent repeatability. It provides a practical tool for early and rapid diagnosis of CIAV, clinical sample screening in grassroots veterinary laboratories, and on-site epidemiological surveillance in poultry farms, holding significant potential for the precise prevention, control, and eradication of CIAV in the poultry industry.