Development and Analytical Evaluation of a Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Seasonal Influenza A Virus Detection

Abstract

Background: Seasonal influenza A virus (IAV) continues to pose a substantial global health threat because of its high mutation rate, widespread prevalence, and the ongoing challenge of timely detection, particularly in resource-limited settings. Objectives: To address this gap, we developed and analytically evaluated a novel colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid, sensitive, and visually interpretable detection of seasonal IAV. Methods: Ten reported LAMP primer sets previously reported were initially screened for amplification efficiency using certified reference RNA materials from H1N1 and H3N2 as templates. Analytical sensitivity was assessed using serial dilutions of quantified RNA standards, and specificity was rigorously evaluated through both in silico analysis and experimental testing with a panel of common respiratory pathogens. Results: Sensitivity analyses based on triplicate serial dilution testing revealed preliminary limits of detection as low as 67 copies per reaction for H1N1 and 42 copies per reaction for H3N2, with performance matching or exceeding that reported in previous LAMP-based studies. No cross-reactivity with other respiratory pathogens was observed, confirming the high specificity of the assay. The assay targets the conserved matrix protein gene and detects IAV broadly; however, it does not differentiate between the H1N1 and H3N2 subtypes in a single reaction. Notably, incorporation of hydroxy naphthol blue (HNB) enabled direct visual detection via a distinct color change from violet to sky blue within 45 minutes under isothermal conditions, thereby eliminating the need for advanced instrumentation. Conclusions: The assay demonstrated high sensitivity and specificity, supporting future clinical validation studies to establish its utility for point-of-care testing (POCT) deployment.