PCR-BASED DETECTION OF AUJESZKY'S DISEASE VIRUS (SUID ALPHAHERPESVIRUS 1) USING EVAGREEN REAL-TIME PCR ASSAY

Abstract

Aujeszky’s disease virus (ADV) poses significant economic and animal health challenges in swine populations. Rapid, sensitive, and specific detection methods are essential for early diagnosis and effective disease control. Study was aimed to develop and validate a real-time PCR assay using EvaGreen chemistry on the iTOWER platform for the detection of the ADV gE gene, evaluating its analytical performance, specificity, reproducibility, and diagnostic sensitivity compared to conventional methods. The assay was tested on clinical samples from 30 swine, with amplification performance assessed through sigmoidal fluorescence curves and melting curve analysis. Analytical sensitivity, limit of detection (LOD), amplification efficiency, linearity (R²), and reproducibility were evaluated using serial dilutions of ADV DNA. Specificity was confirmed using heterologous viral DNA, and diagnostic performance was compared with allergic testing and virus neutralization assays. The developed qPCR assay reliably detected ADV DNA in all clinically positive samples and identified an additional low-viral-load case missed by virus neutralization. Ct values ranged from 18.4 to 33.2, with high reproducibility (coefficient of variation <2%). Melting curve analysis confirmed amplification specificity, with single peaks corresponding to the 235 bp gE amplicon. Analytical sensitivity reached 10² genome copies per reaction, with optimal amplification efficiency (100%) and strong linearity (R² = 0.998) across a five-log dynamic range. No cross-reactivity was observed with non-target herpesviruses. The EvaGreen-based qPCR assay provides a rapid, sensitive, and specific tool for the detection of ADV, suitable for both diagnostic and epidemiological applications. Its integration into One Health surveillance frameworks can enhance early detection and support disease management in swine populations.