Optimization of Loop-Mediated Isothermal Amplification for Avian Influenza Detection

Abstract

Avian influenza viruses (AIV) cause severe economic losses in poultry production and pose zoonotic threats, necessitating rapid, field-deployable diagnostics. While real-time PCR is the gold standard, its use is limited in resource-constrained settings. This study aimed to develop and validate optimized loop-mediated isothermal amplification (LAMP) protocols for AIV detection directly at sample collection sites. We optimized Real-Time RT-LAMP and colorimetric LAMP assays targeting the conserved M gene, using primers described in the literature. Analytical sensitivity was assessed with a plasmid standard (106–100 copies/μL); specificity was evaluated against 27 AIV strains (H1–H12) and heterologous avian viruses (Newcastle disease, infectious bronchitis, Gumboro, and laryngotracheitis viruses). Reverse transcription was integrated into the LAMP reaction. Real-Time LAMP with SYBR Green achieved 100% analytical sensitivity (95% CI: 80–100; detection limit: Ct = 38), while colorimetric LAMP (cresol red, malachite green, calcein) detected 102 plasmid copies (Ct = 32) with 91.67% sensitivity (95% CI: 76.1–100). No cross-reactivity occurred. These optimized LAMP protocols offer sensitivity and specificity comparable to PCR, require minimal equipment, and enable rapid AIV screening, significantly enhancing early detection and epidemiological surveillance in field conditions.