Diagnostic Protocol for Infectious Spleen and Kidney Necrosis Virus (ISKNV) Nucleic Acid Detection in Mandarin Fish

The Infectious Spleen and Kidney Necrosis Virus (ISKNV) was first discovered in Mandarin fish in China in 1997, and cases have since been reported in several Asian countries as well as in Canada, Australia, and Germany. Infected fish may exhibit symptoms such as body imbalance, hypoxia, congestion of the gills and abdomen, protruding eyes, and bleeding from the gill filaments. ISKNV in Mandarin fish is characterized by a rapid onset, high mortality rate, and can lead to mass fish deaths in a short period of time.

I. Purpose

This protocol aims to establish a rapid and accurate nucleic acid detection method for diagnosing Infectious Spleen and Kidney Necrosis Virus (ISKNV) in Mandarin fish to enable timely identification of the virus infection and prevent its spread.

II. Scope of Application

It is applicable for early diagnosis and monitoring of ISKNV infection in Mandarin fish and other freshwater fish by aquaculture farms, research institutions, and relevant departments.

III. Detection Principle

Polymerase Chain Reaction (PCR) technology is used to amplify specific gene segments of ISKNV through specific primers, enabling the detection of the virus nucleic acid.

IV. Sample Collection and Processing

1. Sample Collection: Collect samples from the suspected infected spleen, kidney, or liver tissues of Mandarin fish.
2. Sample Processing: Grind the tissue samples and extract the viral nucleic acid using appropriate buffers.

V. Main Reagents and Instruments

• PCR primers
• DNA polymerase
• dNTPs
• Nucleic acid extraction reagent kit
• PCR machine
• Microcentrifuge
• Agarose gel electrophoresis system

VI. Experimental Steps

1. Nucleic Acid Extraction: Follow the instructions of the nucleic acid extraction kit to extract the viral nucleic acid from the samples.
2. PCR Amplification: Design specific primers, set up the PCR reaction system, and carry out amplification.
3. Electrophoresis Analysis: Perform agarose gel electrophoresis on the PCR products to observe specific bands.

VII. Result Interpretation

Based on the electrophoresis results after PCR amplification, the appearance of the expected size of specific bands indicates ISKNV positivity; absence of specific bands or mismatch in band size indicates negativity.

VIII. Precautions

• Conduct experiments in a biosafety cabinet to prevent cross-contamination.
• Properly label all reagents and samples to avoid confusion.
• Strictly follow the operational procedures to ensure result accuracy.

IX. Quality Control

Regularly conduct positive and negative control experiments to validate the reliability of the detection method.

X. Report Preparation

After completion of the detection, promptly prepare a detection report with sample information, results, operators, etc., and archive it for reference.