Indian Journal of Animal Research
SCOPUSWeb of Science
  • Year: 2026
  • Volume: 60
  • Issue: 2

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

  • Author:
  • Alagukanthasami Ponsrinivasan1*, Arumugam Uma1, P. Chidambaram2, Cheryl Antony2, Arun Sudhagar3, Sethu Selvaraj4, S. Ganesh Babu5
  • Total Page Count: 8
  • Page Number: 262 to 269

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

Abstract

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.

Keywords

Aquaculture diagnostics, Multiplex PCR, Tilapia lake virus, Viral detection