Development and comparative evaluation of real-time PCR and colorimetric loop-mediated isothermal amplification (LAMP) assays for the detection of infectious spleen and kidney necrosis virus (ISKNV) in tilapia

Abstract

Infectious Spleen and Kidney Necrosis Virus (ISKNV) is a highly lethal pathogen posing a major threat to global aquaculture. The recent emergence of ISKNV on the African continent has had devastating impacts on the tilapia fish industry. There is a critical need for rapid, sensitive, and field-adaptable diagnostic tools suited to local conditions. In this study, a SYBR green-based real-time quantitative PCR (qPCR) was developed and evaluated alongside an adapted colorimetric loop-mediated isothermal amplification (LAMP) assay. The sensitivity, specificity, precision, and diagnostic applicability of the two methods were compared against a reference multiplex conventional PCR (mPCR). Both assays were specific for ISKNV detection in infected BF-2 cell lines and fish tissues samples. The LAMP assay demonstrated the highest analytical sensitivity, with a detection limit (LOD) of approximately 17 viral copies/µL under the experimental conditions used in this study. The assay also enabled rapid detection from crude tissue homogenates without prior nucleic acid extraction, indicating its potential utilization for field-based diagnostics. The qPCR assay achieved ten-fold lower sensitivity with an LOD of 67 viral copies/µL, which is still sensitive enough to support its use in laboratory-based diagnostics and epidemiological surveillance during outbreak investigations. Collectively, these assays provide complementary molecular tools to support ISKNV detection and surveillance in resource-limited settings, pending broader diagnostic validation.