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. 2025 Apr 9;15(1):12110.
doi: 10.1038/s41598-025-95452-6.

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

Mohamed Kamel  1   2 , Josiah Levi Davidson  1 , Jenna M Schober  3 , Gregory S Fraley  3 , Mohit S Verma  4   5   6
Affiliations

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

Mohamed Kamel et al. Sci Rep. .

Abstract

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.

Keywords: Diagnostics; Influenza A viruses; Loop-mediated isothermal amplification; Microfluidic paper-based analytical devices; Point-of-care; µPADs.

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Conflict of interest statement

Declarations. Competing interests: M.S.V. has interests in Krishi Inc., which is a startup company developing molecular assays. Krishi Inc. did not fund this work. Remaining authors do not have a competing interest.

Figures

Fig. 1
Fig. 1
Screening of LAMP primer sets for H5 subtype AIV by fluorescent LAMP assay using the NEB Warm Start DNA/RNA LAMP Kit.
Fig. 2
Fig. 2
LOD experiment showing the result of the H5 subtype AIV qLAMP assay of the HPAIV.HA.5 primer set using the fluorescent qLAMP assay testing different concentrations of IVT RNA (18900, 1890, 500, 250, 125, 50, 25, 5, and 1 copies/reaction).
Fig. 3
Fig. 3
Limit of Detection (LOD) experiment of the H5 subtype AIV colorimetric liquid LAMP assay using the HPAIV.HA.5 primer set. The colorimetric liquid LAMP assay was performed to test different concentrations of synthetic RNA (1890, 500, 250, 125, 50, 25, 5, and 1 copies/reaction). The figure shows a LOD of 500 copies/reaction for the H5 subtype AIV colorimetric liquid LAMP assay using the HPAIV.HA.5 primer set, where the color changes from pink to yellow.
Fig. 4
Fig. 4
Paper-based LAMP LOD: Limit of Detection (LOD) experiment of the H5 subtype AIV colorimetric paper-based LAMP assay using the HPAIV.HA.5 primer set. The colorimetric paper-based LAMP assay was performed to test different concentrations of synthetic RNA.
Fig. 5
Fig. 5
Schematic and colorimetric evaluation of the paper-based device. (A) Step-by-step workflow diagram. The no-primer control is indicated by the control zone. (B) Schematic representation of the paper device layout. (C) Colorimetric readout obtained from negative and positive tests.
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