A paper-based loop-mediated isothermal amplification assay for highly pathogenic avian influenza

Avian influenza outbreaks have had significant economic and public health consequences worldwide. Therefore, prompt, reliable, and cost-effective diagnostic devices are crucial for scrutinizing and confining highly pathogenic avian influenza viruses (HPAIVs). Our study introduced and evaluated a novel paper-based loop-mediated isothermal amplification (LAMP) test for diagnosing the H5 subtype of the avian influenza virus (AIV). We meticulously designed and screened LAMP primers targeting the H5-haemagglutinin (H5-HA) gene of AIV and fine-tuned the paper-based detection assay for best performance. The paper-based LAMP assay demonstrated a detection limit of 500 copies per reaction (25 copies/µl). Additionally, the assay exhibited no cross-reactivity with common bovine and avian pathogens, confirming its specificity. Spiking experiments revealed that the assay could accurately detect 1000 copies of synthetic HPAIV RNA (per reaction) when spiked into oropharyngeal swab samples, achieving 100% analytical sensitivity, specificity, and accuracy. This inexpensive, user-friendly point-of-need diagnostic tool holds great promise, especially in resource-limited settings. It only requires a water bath for incubation and enables visual detection of results without special equipment. Overall, the paper-based LAMP assay provides a promising method for rapidly and reliably detecting the H5 subtype of AIV, contributing to improved surveillance and early intervention strategies.