Development and clinical application of a TaqMan-based real-time quantitative PCR assay for rapid detection of goose adenovirus type 4

Abstract

Introduction Goose adenovirus type 4 (GoAdV-4) is an emerging pathogen that causes inclusion body hepatitis and hepatic necrosis in goslings, with mortality rates reaching up to 80% in severe outbreaks. Since its first identification in China in 2022, GoAdV-4 has spread rapidly across major goose-producing provinces, posing a serious threat to the domestic goose industry. However, no sensitive and specific quantitative assay has been available for the rapid diagnosis and viral load monitoring of GoAdV-4. Methods We developed a TaqMan-based real-time quantitative PCR (qPCR) assay targeting the hexon gene of GoAdV-4 and systematically validated its analytical sensitivity, specificity, and repeatability. The assay was then applied to 582 clinical samples of five specimen types (tissues, blood, goose embryos, and cloacal swabs), and its diagnostic performance was compared with conventional PCR. Results The assay demonstrated a linear detection range of 6.4 × 101 to 6.4 × 107 copies/μL (R2 = 0.9968) with an amplification efficiency of 97.5%, and a limit of detection (LOD) as low as 6.4 × 101 copies/μL. No cross-reactivity was observed with fowl adenovirus serotype 4, duck adenovirus type 3, egg drop syndrome virus, goose circovirus, goose astrovirus, goose parvovirus, or nuclease-free water. Intra-assay and inter-assay coefficients of variation ranged from 0.87% to 1.17% and 1.06% to 1.48%, respectively, indicating high reproducibility. The assay identified 55 GoAdV-4-positive samples (9.45%), compared with 48 positives (8.25%) detected by conventional PCR, with an overall concordance rate of 98.80% (positive concordance 87.27%, negative concordance 100%). Discussion The TaqMan qPCR assay detected seven additional positive samples missed by conventional PCR, all confirmed as true positives by Sanger sequencing, demonstrating superior sensitivity. These results show that the established TaqMan qPCR assay is a rapid, sensitive, specific, and reproducible tool for GoAdV-4 detection, providing a valuable diagnostic instrument for the surveillance and control of this emerging waterfowl pathogen.