A Triplex Crystal Digital RT-PCR for the Detection of Avian Leukosis Virus, Chicken Infectious Anemia Virus, and Fowl Adenovirus.

Avian leukosis virus (ALV), chicken infectious anemia virus (CIAV), and fowl adenovirus (FAdV) are significant immunosuppressive agents that cause huge financial losses to the poultry industry. In this study, specific primers and probes for ALV, CIAV, and FAdV were designed, and the RNA standard for ALV and plasmid standards for CIAV and FAdV were constructed. A triplex reverse transcription-crystal digital PCR (RT-cdPCR) assay was developed for the detection of ALV, CIAV, and FAdV after the optimization of key reaction parameters, including primer and probe concentrations, annealing temperature, and reaction cycle. Subsequently, the assay was assessed for its specificity, sensitivity, and repeatability. The results showed that the assay specifically detected ALV, CIAV, and FAdV, without cross-reactivity with other avian pathogens. The limits of detection (LODs) for sensitivity to ALV, CIAV, and FAdV were 3.75, 3.75, and 5.50 copies/reaction, respectively. Repeatability analysis showed the coefficients of variation (CVs) with 0.38-1.77% for intra-assay and 0.31-3.02% for inter-assay. The assay was applied to evaluate 1211 clinical samples from Guangxi Zhuang Autonomous Region in China and yielded positivity rates of 45.58% (552/1211) for ALV, 10.73% (130/1211) for CIAV, and 5.20% (63/1211) for FAdV. Additionally, these 1211 clinical samples were tested using the reference methods, and the results were compared with those of the established method, showing coincidence rates ≥ 98.18%. In conclusion, a triplex RT-cdPCR was successfully established for the simultaneous and accurate detection of ALV, CIAV, and FAdV.