Related Experiment Video
Updated: Jun 11, 2026
03:53
Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV
Published on: November 10, 2023
Development of a multiplex real-time PCR method for detecting immunosuppressive viruses and its preliminary
Zhenyu Chen1, Yufu Li2, Yiyang Huang1
1College of Life Engineering, Shenyang Institute of Technology, Fushun 113122, China.
Journal of Virological Methods
|June 9, 2026
Summary
A new multiplex real-time qPCR assay effectively detects six common immunosuppressive viruses in broilers. Chicken anemia virus (CAV) was most prevalent, with frequent mixed infections observed in Liaoning Province flocks.
Area of Science:
- Veterinary Virology
- Molecular Diagnostics
- Avian Pathology
Background:
- Six prevalent immunosuppressive viruses (MDV, CAV, FADV, REV, ARV, IBDV) significantly impact broiler health and productivity.
- These viral infections often occur concurrently, complicating diagnosis and disease management.
- Existing diagnostic methods may not efficiently detect multiple viral agents simultaneously.
Purpose of the Study:
- To develop and validate a multiplex real-time quantitative PCR (qPCR) assay for simultaneous detection of six key broiler immunosuppressive viruses.
- To assess the prevalence and co-infection patterns of these viruses in clinical samples from diseased chickens.
Main Methods:
- Design of specific primers and TaqMan probes targeting conserved genes of 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).
- Establishment of a one-step RT-qPCR protocol for high-efficiency, sensitive, and stable detection.
- Validation of the assay's analytical sensitivity (limit of detection), precision (coefficient of variation), linearity (R²), and amplification efficiency.
- Application of the developed assay to 94 clinical liver samples from broiler flocks.
Main Results:
- The multiplex qPCR assay demonstrated high efficiency, stability, and sensitivity, with a minimum detectable plasmid level of 1 copy/µL for all targets.
- Excellent linearity (R² ≥ 0.998) and amplification efficiency (96%-104%) were achieved for all six viral detections.
- Chicken anemia virus (CAV) showed the highest positivity rate (91.49%) in clinical samples.
- Significant rates of co-infections were observed: 30.00% dual, 37.78% triple, 13.33% quadruple, and 1.11% quintuple infections.
Conclusions:
- The developed multiplex real-time qPCR method provides a novel, efficient, and reliable tool for diagnosing multiple immunosuppressive viral infections in broilers.
- High prevalence and frequent co-infections with MDV, CAV, FADV, REV, ARV, and IBDV are significant issues in broiler flocks in Liaoning Province.
- This diagnostic approach aids in understanding the complex etiology of immunosuppressive diseases in poultry and informs targeted disease control strategies.
