Rapid, non-invasive, visual point-of-care detection of koi herpesvirus using loop-mediated isothermal amplification

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Abstract
Introduction: Koi herpesvirus (KHV or CyHV-3) causes a highly contagious and lethal disease in common carp and koi, resulting in substantial economic losses in global aquaculture. Rapid point-of-care testing (POCT) of KHV is critical for curbing its spread and preventing outbreaks. However, conventional diagnostic approaches, such as TaqMan quantitative PCR (qPCR), require professional personnel and specialized equipment, limiting their on-site applicability. Methods: A rapid visual POCT assay was developed by combing loop-mediated isothermal amplification (LAMP) with non-invasive mucus swab sampling. Two optimized release solutions enabled one-step nucleic acid preparation within 5 min. Among five primer sets designed, the optimal set targeting the thymidine kinase (TK) gene was selected for isothermal amplification at 65 °C for 60 min. The assay was further adapted for naked-eye result interpretation using a pH-sensitive indicatior dye. Results: The assay demonstrated a detection limit of 21.42 copies/μL, with a 95% confidence interval of 14.88—41.83 copies/μL. No cross-reactivity was observed with seven common fish pathogens. Comparative evaluation with TaqMan qPCR using parallel clinical specimens yielded 100% concordant results. The total detection time was 65 min, enabling the POCT implementation across diverse scenarios. Discussion: This non-invasive, visual LAMP-based POCT platform provides a sensitive, specific and equipment-free diagnostic strategy for KHV detection, facilitating timely disease surveillance, outbreak control management, and aquaculture biosecurity.
Funding Information
  • Beijing Academy of Agricultural and Forestry Sciences
  • Guiyang University

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