RT-LAMP as Diagnostic Tool for Influenza-A Virus Detection in Swine

Area of Science:

• Veterinary Diagnostics
• Molecular Virology
• Point-of-Care Testing

Background:

• Point-of-care diagnostic technologies are increasingly vital for production animal health.
• Influenza A virus in swine (IAV-S) poses significant risks to animal agriculture.
• Rapid and accurate detection methods are crucial for controlling IAV-S outbreaks.

Purpose of the Study:

• To develop and evaluate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting the matrix (M) gene of IAV-S.
• To assess the sensitivity, specificity, and limit of detection (LOD) of the developed RT-LAMP assay.
• To determine the potential of the RT-LAMP assay as a rapid, low-cost screening tool for IAV-S.

Main Methods:

• Design of M gene-specific LAMP primers based on recent IAV-S sequences from the USA.
• Optimization of the RT-LAMP assay conditions, including incubation temperature (65 °C) and time (30 min).
• Evaluation of the assay's LOD using direct gene standards, spiked extraction kits, and cell culture samples.
• Validation of the assay's performance using clinical swine samples, assessing sensitivity and specificity.

Main Results:

• The RT-LAMP assay demonstrated a limit of detection of 20 M gene copies for direct standards.
• The LOD was 100 M gene copies with spiked extraction kits and 1000 M genes in cell culture samples.
• Clinical sample analysis yielded a sensitivity of 94.3% and a specificity of 94.9%.
• The assay successfully detected the influenza M gene under research laboratory conditions.

Conclusions:

• The developed RT-LAMP assay is a viable method for detecting IAV-S.
• The assay exhibits high sensitivity and specificity, comparable to established diagnostic methods.
• With appropriate equipment, this RT-LAMP assay can be rapidly validated as a low-cost, on-farm or clinical screening tool for IAV-S, facilitating timely disease management.