Development of a multiplex real-time PCR method for detecting immunosuppressive viruses and its preliminary application in broilers.

Marek's disease virus (MDV), Chicken anemia virus (CAV), fowl adenovirus (FADV), avian reticuloendotheliosis virus (REV), avian reovirus (ARV), and infectious bursal disease virus (IBDV) are prevalent immunosuppressive diseases affecting broilers, exhibiting a high prevalence rate.To address this issue, a multiplex real-time quantitative PCR(qPCR) method was designed for the detection of all six viruses. The study involved designing primers and probes that specifically target the MDV Meq gene, CAV VP2 gene, FADV penton base gene, REV gp90 gene, ARV S1 gene, and IBDV VP2 gene, and it established a TaqMan probe-based one-step RT-qPCR method.This method is characterized by high efficiency, stability, and sensitivity, achieving a minimum detectable plasmid level of 1 copy/µL for all six viruses. Furthermore, the coefficient of variation for all six viral detection methods is below 3%. The method demonstrates a strong linear relationship (R² ≥ 0.998) and an amplification efficiency ranging from 96% to 104%. Testing of 94 clinical liver samples from diseased chickens indicated that CAV exhibited the highest positivity rate at 91.49%. Among the positive samples, the rates of dual, triple, quadruple, and quintuple mixed infections were 30.00%, 37.78%, 13.33%, and 1.11%, respectively.Research indicates that the qPCR method represents a novel approach for detecting six types of immunosuppressive disease viruses in broilers. These viruses demonstrate a high positive rate and co-infection phenomena among chicken flocks in Liaoning Province.