Development and validation of a multiplex TaqMan real-time quantitative polymerase chain reaction assay for selected...

Accurate quantification of host immune gene expression is essential for elucidating host-pathogen interactions and inflammation regulation in poultry infection studies. However, many existing chicken immune gene-expression assays rely on singleplex reactions or intercalating-dye chemistry, which may limit throughput and target specificity. Here, we developed and validated a probe-based multiplex TaqMan real-time qPCR assay for simultaneous quantification of four selected chicken pro-inflammatory immune-response transcripts: IFN-γ, IL-1β, IL-8L1, and LITAF, with 18S rRNA included as a separately quantified reference target. RNA quantity and purity were assessed spectrophotometrically, and RNA integrity was evaluated by agarose gel electrophoresis. Plasmid DNA standard curves demonstrated amplification efficiencies within 90-110% and strong linearity with R² ≥ 0.98 in both singleplex and multiplex formats, while RT-derived biological cDNA dilution series confirmed target-specific linearity within biological template matrices. The plasmid-based standard curves covered an approximate Ct range of 15-32, supporting quantification across the validated linear dynamic range. Lung samples collected at 1 and 3 days post-infection from an established avian pathogenic Escherichia coli (APEC) challenge study were selected for proof-of-concept testing based on prior SYBR Green qPCR results. The assay detected significant upregulation of IL-1β and IL-8L1 at 1 day post-infection, with no significant changes in IFN-γ and LITAF. Follow-up SYBR-based checks supported the same interpretation and argued against chemistry- or primer-set artifacts. This focused multiplex assay provides a practical research tool for simultaneous measurement of selected chicken inflammatory immune-response transcripts from limited template input. Its application was demonstrated in APEC-infected lung tissue, and additional tissue-, pathogen-, and reference-gene-specific validation will be required before broader application.