Comparative Evaluation of IndiMix JOE with intype IC-RNA as an Alternative to AgPath-ID for Influenza A Virus Detection Across Avian and Bovine Matrices

This study evaluated IndiMix JOE with intype IC-RNA as an alternative PCR chemistry to the National Animal Health Laboratory Network (NAHLN) Influenza A virus (IAV) PCR surveillance assay using AgPath-ID One Step PCR reagents in avian swabs and tissues and bovine milk. In avian samples, IndiMix JOE with intype IC-RNA using a fast reduced-volume (FRV, 20 µL) protocol had comparable results to NAHLN standard PCR assays (with and without intype IC) using standard NAHLN thermocycling conditions (25 µL). Precision was high, with coefficients of variation ≤ 2.61% for IAV targets and ≤ 2.03% for intype IC-RNA. Following detection of IAV in U.S. dairy cattle, a six-way comparison of PCR chemistries and exogenous internal controls in milk illustrated no significant differences in mean CT values (ANOVA, p = 0.9938). Additional experiments in avian, milk and semen samples were performed, resulting in comparable analytical sensitivity in limits of detection (LOD), linearity (R² > 0.977), and PCR efficiencies, and lacked significant differences in mean CT values (ANOVA, p > 0.05). Diagnostic sensitivity and specificity were 100% across matrices. These findings validate IndiMix JOE and intype IC-RNA as a reliable alternative reagents that enhances testing flexibility, efficiency, and outbreak response capacity.