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ARS Home » Southeast Area » Mississippi State, Mississippi » Poultry Research » Research » Publications at this Location » Publication #432499
Research Project: Intervention Strategies to Mitigate Avian Escherichia coli Infections and Antimicrobial Resistance in the Poultry Environment

Location: Poultry Research

Title: Development and validation of a multiplex TaqMan real-time quantitative polymerase chain reaction assay for selected pro-inflammatory immune-response gene expression in chickens

Author
item YE, XIN - Mississippi State University
item HSU, CHUAN-YU - Mississippi State University
item JIA, LINAN - Mississippi State University
item Magee, Christopher
item Leigh, Spencer
item Evans, Jeffrey
item ZHANG, LI - Mississippi State University
item Robinson, Kelsy

Submitted to: The Journal of Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/8/2026
Publication Date: 6/24/2026
Citation: Ye, X., Hsu, C., Jia, L., Magee, C.L., Leigh, S.A., Evans, J.D., Zhang, L., Robinson, K. 2026. Development and validation of a multiplex TaqMan real-time quantitative polymerase chain reaction assay for selected pro-inflammatory immune-response gene expression in chickens. The Journal of Poultry Science. 105: 107237. https://doi.org/10.1016/j.psj.2026.107237.
DOI: https://doi.org/10.1016/j.psj.2026.107237

Interpretive Summary: Understanding how the immune system responds during infection is a key element of developing new, more effective approaches to prevent and treat disease. Measurement of gene expression is one of the most common methods used to evaluate immune response. However, the current techniques can only measure one gene at a time making them slow and less precise. To advance our ability to characterize host response, we developed an assay that can measure four important immune signals all at once. When tested in both lab and animal models, the results showed the assay to be accurate and reliable. In an animal infection model, the assay was able to accurately detect rapid changes in lung cytokine expression at only one day post-infection. These results show that this assay can provide an accurate survey of immune function while saving time, sample, and reagents to help scientists get results faster.

Technical Abstract: Accurate and standardized quantification of host cytokine transcript expression is essential for elucidating host–pathogen interactions and immune regulation in poultry infection and inflammation studies. However, many existing avian cytokine assays rely on singleplex reactions or intercalating-dye chemistry, which can limit throughput and specificity. Here, we developed and validated a probe-based multiplex TaqMan qPCR panel for simultaneous quantification of four host pro-inflammatory cytokine transcripts (IFN-', IL-1ß, IL-8L1, and TNF-a), with 18S rRNA included as a reference target. Standard curves constructed using plasmid DNA standards and RT-derived biological cDNA templates demonstrated high efficiency (90–110%) and strong linearity (R² = 0.98) across targets, supporting robust quantitative performance in both singleplex and multiplex formats. As a proof-of-concept application, the panel was applied to lung tissues from an avian pathogenic Escherichia coli (APEC) challenge model, where IL-1ß and IL-8L1 were upregulated at 1 day post-infection, whereas IFN-' and TNF-a showed no significant changes. Cross-chemistry and primer-set reanalysis of discrepant comparisons supported concordant inference and argued against chemistry- or primer/amplicon-driven artifacts. This multiplex qPCR system provides a rapid, sensitive, and target-specific approach for multi-gene immune profiling from limited template input and offers a transferable framework that can be deployed as a two-, three-, or four-plex and adapted to additional panels and tissues for standardized immune monitoring in avian systems.