Multiplex PCR Targeting Multiple Genome Segments for Enhanced Detection of Tilapia Lake Virus (TiLV) in Tilapia

Background: Tilapia Lake Virus (TiLV) is an emerging threat to global aquaculture and current PCR assays that target a single genome segment is vulnerable to false negatives due to genetic variability and viral reassortment. A robust diagnostic tool is needed for reliable detection and surveillance. Methods: We developed a multiplex polymerase chain reaction (mPCR) assay targeting three conserved genome segments (2, 3 and 8) using TiLV Primer sets that were optimized through uniplex and multiplex reactions and assay conditions were standardized at 58°C. Sensitivity was assessed using serial dilutions of TiLV-positive cDNA, while specificity was evaluated against non-TiLV aquatic pathogens. Diagnostic performance was compared with semi-nested reverse transcription polymerase chain reaction (semi-nested RT-PCR) and validated using Receiver Operating Characteristic (ROC) analysis. Result: The optimized mPCR assay consistently amplified all three segments in a single reaction, producing clear and specific bands without non-specific amplification. The detection limit was 100 (picogram) pg/µL of TiLV cDNA and no cross-reactivity was observed with non-TiLV pathogens. ROC analysis yielded an AUC value of 1.0, indicating perfect sensitivity and specificity. This multi-segment approach minimizes false negatives and offers a reliable tool for TiLV detection in tilapia aquaculture.