Development of a loop-mediated isothermal amplification (LAMP) assay for rapid visual detection of snakehead vesiculovirus (SHVV) in snakehead

Abstract

Infections caused by snakehead vesiculovirus (SHVV) have seen frequent outbreaks in recent years, inflicting significant losses on the snakehead aquaculture industry. Early detection is therefore essential for effective prevention and control of pathogenic infections and reduction of economic losses caused by infections. There is an urgent need for a simple, rapid, specific, sensitive, and intuitive method to monitor snakehead infected with SHVV. The aim of the present study was to develop and evaluate a loop-mediated isothermal amplification (LAMP) assay for the rapid visual detection of SHVV in snakehead. Three pairs of primers were designed according to the conserved region of phosphoprotein (P) gene sequences of SHVV and were applied for the detection of SHVV from fish samples. Time and temperature conditions for the amplification of SHVV were optimized at 65 °C and 55 min. The LAMP assay demonstrated high specificity, with no cross-reactivity with seven other viruses. Amplification results were visualized by a color change after the addition of hydroxynaphthol blue (HNB) dye. Sensitivity test results showed that the minimum detection volume with this method was 1.76 × 10² copies/μL, which was 100 times more sensitive than RT-PCR assay. We used the established LAMP system to test 50 clinical samples and detected 32 positive responses, whereas 22 positive samples out of 50 samples were detected by RT-PCR. The establishment of a visual LAMP assay further shortens the virus detection process and allows visual reading of positive responses through color changes; it is suitable for use in quarantine and field detection. Therefore, this proposed method provides a sensitive, specific, and user-friendly method for the rapid diagnosis of SHVV in snakehead farming.