Point-of-Care Diagnostic Modular Device for Detection of VHSV with Reverse Transcription Loop-Mediated Isothermal Amplification

Abstract A portable RT-LAMP assay was developed for the rapid and sensitive detection of viral hemorrhagic septicemia virus (VHSV), addressing the limitations of traditional PCR-based diagnostics in field settings, especially in aquaculture. The existing method based on conventional PCR is hindered by the immobility of the equipment, making early detection and response in remote environments challenging. To overcome this, the developed RT-LAMP system integrates modular components, including fluid connections and a heating block that ensures precise temperature control with deviations within ± 0.1℃, enabling reliable amplification. Optimized primers targeting the VHSV N gene, specifically the N37 set, demonstrated high sensitivity, detecting viral loads as low as 56 aM (34 copies/µl). The assay was further validated for specificity using a broad panel of controls, including RSIV, influenza A/B, ASFV, and Shigella , with no cross-reactivity observed. Field trials using POCT modules achieved 94.8% sensitivity and 100.0% specificity. This system enables rapid and accurate diagnostics in resource-limited environments, facilitating the early detection of VHSV in aquaculture, and has potential for automation, integrating sample processing and detection for broader diagnostic applications.