Development and preliminary application of a multiplex qPCR assay for the detection of avian leukosis virus subgroup J, reticuloendotheliosis virus, and chicken infectious anemia virus

Avian leukosis virus subgroup J (ALV-J), Reticuloendotheliosis virus (REV), and Chicken infectious anemia virus (CIAV) are important breeder-derived pathogens (BDPs) that continue to threaten poultry health and productivity. These viruses are frequently associated with immunosuppression and coinfections, which complicate eradication efforts, particularly in indigenous chicken breeds. In this study, a TaqMan-based multiplex qPCR (M-qPCR) assay was developed for the simultaneous detection of ALV-J, REV, and CIAV. The assay exhibited excellent linearity (R² > 0.99), high repeatability (intra-/inter-assay coefficient of variation < 5%), strong specificity with no cross-reactivity against common non-target avian pathogens, and high sensitivity with a detection limit of 10 copies/μL. Moreover, it reliably identified mixed infections even under conditions of unbalanced target concentrations. Parallel testing with commercial singleplex qPCR kits further demonstrated its reliability, yielding concordance rates of 95.8% for ALV-J, 98.3% for REV, and 100% for CIAV. Application of the assay to three local chicken breeds in Guizhou revealed positivity rates of 33.9% (40/118) for ALV-J, 9.3% (11/118) for REV, and 7.6% (9/118) for CIAV, with 20.4% mixed (double and triple) out of all positives. Moreover, serum samples proved more suitable than cloacal swabs for virus detection using the developed M-qPCR assay. In conclusion, the developed M-qPCR assay provides a reliable and efficient tool for surveillance of infections and coinfections in poultry flocks, supporting the prevention and control of BDPs.