1Department of Aquatic Animal Health Management, Dr. M.G.R. Fisheries College and Research Institute, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Ponneri-601 204, Tamil Nadu, India.
2Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Vettar River View Campus, Nagapattinam-611 002, Tamil Nadu, India.
3Centre for Peninsular Aquatic Genetic Resources, ICAR-National Bureau of Fish Genetic Resources, Kochi-682 018, Kerala, India.
4Department of Aquaculture, Dr. M.G.R. Fisheries College and Research Institute, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Ponneri-601 204, Tamil Nadu, India.
5Department of Basic Sciences, Institute of Fisheries Post Graduate Studies, Tamil Nadu Dr. J. Jayalalithaa Fisheries University OMR Campus, Vaniyanchavadi, Chennai-603 103, Tamil Nadu, India.
*Corresponding Author: Alagukanthasami Ponsrinivasan, Department of Aquatic Animal Health Management, Dr. M.G.R. Fisheries College and Research Institute, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Ponneri-601 204, Tamil Nadu, India. Email: ponsrinivasan97@gmail.com
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.
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.
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.
Aquaculture diagnostics, Multiplex PCR, Tilapia lake virus, Viral detection
