Optimization of Loop-Mediated Isothermal Amplification for Avian Influenza Detection

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Public Health

Background:

• Avian influenza viruses (AIV) cause significant economic losses in poultry and pose zoonotic risks.
• Current gold-standard diagnostics like real-time PCR are often impractical in resource-limited field settings.
• Rapid, on-site diagnostic tools are crucial for effective AIV surveillance and control.

Purpose of the Study:

• To develop and validate optimized loop-mediated isothermal amplification (LAMP) protocols for the direct detection of AIV at sample collection sites.
• To integrate reverse transcription into the LAMP reaction for a streamlined workflow.
• To assess the analytical sensitivity and specificity of the developed LAMP assays.

Main Methods:

• Optimization of Real-Time RT-LAMP and colorimetric LAMP assays targeting the conserved M gene of AIV.
• Use of pre-existing primer sets from scientific literature.
• Analytical sensitivity testing using a plasmid standard across a range of concentrations (10^6-10^0 copies/μL).
• Specificity evaluation against a panel of 27 AIV strains (H1-H12) and common heterologous avian viruses.

Main Results:

• Real-Time LAMP with SYBR Green demonstrated 100% analytical sensitivity (detection limit: Ct = 38).
• Colorimetric LAMP assays (using cresol red, malachite green, or calcein) achieved 91.67% sensitivity (detection limit: 10^2 plasmid copies, Ct = 32).
• Both optimized LAMP assays exhibited no cross-reactivity with tested heterologous avian viruses, confirming high specificity.

Conclusions:

• Optimized LAMP protocols provide a sensitive and specific alternative to PCR for AIV detection in field conditions.
• These assays require minimal equipment, enabling rapid screening at sample collection sites.
• The developed LAMP methods significantly enhance early AIV detection and epidemiological surveillance capabilities.