Novel Ultrafast Sample-to-Answer Microfluidic Platform for Highly Sensitive On-Site Quantification of Avian Influenza Virus

Avian influenza virus (AIV) poses a global biosecurity threat, highlighting the need for rapid, accurate, and adaptable diagnostic tools to address its genetic diversity and complex transmission environments. Here, we report the development of FIGHTER, a novel ultrafast sample-to-answer microfluidic platform for the highly sensitive on-site detection of H9 and N2 genes to diagnose the H9N2 AIV subtype. The portable FIGHTER platform integrates an all-in-one plastic-aluminum membrane hybrid microfluidic chip with a dual-channel real-time PCR instrument, enabling on-site nucleic acid extraction in 5 min and reverse transcription-quantitative polymerase chain reaction with ultrafast thermal cycling in 18 min. In addition, the limit of detection of the platform is 1 copy/μL for both H9 and N2. The microfluidic chip with preloaded reagents supports simplified manual operation, and the battery-powered instrument features a user-friendly interface, allowing nonspecialists to operate it in resource-limited settings. Moreover, the validation with 60 field clinical samples showed a clinical sensitivity of 97.92% for H9 and 100% for N2, with 100% specificity for both H9 and N2. Hence, FIGHTER offers a simple, affordable, and rapid method for pathogen detection, particularly in complex environments where the conventional laboratory infrastructure is inaccessible.