Development of a quadruplex RT-qPCR for the detection of avian leukosis virus, chicken infectious anemia virus, avian reovirus, and fowl adenovirus

Avian leukosis virus (ALV), chicken infectious anemia virus (CIAV), avian reovirus (ARV), and fowl adenovirus (FAdV) are important viral pathogens that can transmitted horizontally and vertically, and induce immunosuppression to the poultry flocks. They exhibit diverse pathogenic characteristics in clinical settings, and pose continuous threat to the health of poultry flocks. Here, the specific primers and probes were designed for the env gene of ALV, the VP1 gene of CIAV, the M1 gene of ARV, and the ORF1 gene of FAdV. The RNA standards for ALV, and ARV, and the plasmid DNA standards for CIAV, and FAdV were constructed. To establish a quadruplex real-time quantitative PCR (RT-qPCR) for detecting these four viruses, the reaction conditions (primer and probe concentrations, annealing temperature, and reaction cycles) were optimized, and the specificity, sensitivity, and repeatability were analyzed. The results indicated that the developed assay could specifically detect ALV, CIAV, ARV, and FAdV, and had no cross-reaction with other chicken viruses; the limits of detection (LODs) of them were 136.66, 129.59, 133.20, and 139.79 copies/reaction, respectively, demonstrating high specificity and sensitivity. In addition, this assay had excellent repeatability, with coefficients of variation (CVs) of 0.29-0.93% for the intra-assay and of 0.29-0.99% for the inter-assay. The developed assay was validated via testing 1,575 clinical samples from Guangxi province, China. The positivity rates of ALV, CIAV, ARV, and FAdV were 36.89% (581/1,575), 17.65% (278/1,575), 2.16% (34/1,575), and 7.05% (111/1,575), respectively. These 1,575 clinical samples were also tested using the reported reference methods, and the results were compared with those of the established method. The coincidence rate of the developed and the reference assays exceeded 99.31%. In conclusion, a quadruplex RT-qPCR was successfully developed for the efficient and precise detection and differentiation of ALV, CIAV, ARV, and FAdV.