Comparative analysis of RT-qPCR assay sensitivity and process limit of detection for Japanese encephalitis virus (JEV) detection in piggery wastewater

Japanese encephalitis virus (JEV) poses a significant public health threat in Asia, the Western Pacific, and Australia, necessitating robust surveillance and management strategies. This study evaluates three RT-qPCR assays (Universal JEV, JEV G4, and VIDRL2 JEV G4) for detecting JEV in piggery wastewater, a promising approach for early outbreak detection. We assessed assay limit of detection (ALOD), process limit of detection (PLOD), and recovery efficiency using gamma-irradiated JEV seeded into wastewater samples, alongside field-derived samples from an Australian piggery. The JEV G4 assay demonstrated superior sensitivity, with an ALOD of 2.2–5.7 copies/reaction and PLOD of 72–282 copies/10 mL of piggery wastewater, detecting JEV in 24/30 field samples compared to 17/30 for Universal JEV and 0/30 for VIDRL2 JEV G4. Recovery efficiencies varied, with JEV G4 showing consistent performance (14.9–26.6 %) across concentrations. McNemar's test confirmed JEV G4’s higher sensitivity (p < 0.05). Based on the results obtained in this study, the JEV G4 assay is recommended for wastewater surveillance in genotype 4 regions, with a dual-assay approach suggested for broader genotype coverage. These findings enhance JEV surveillance strategies, supporting early detection and control.