# Optimization of Loop-Mediated Isothermal Amplification for Avian Influenza Detection

## TL;DR

This study develops and validates a rapid, low-tech method for detecting avian influenza viruses in poultry, suitable for use in resource-limited settings.

## Contribution

The paper introduces optimized LAMP protocols for AIV detection with sensitivity and specificity comparable to PCR, suitable for field use.

## Key findings

- Real-Time LAMP with SYBR Green achieved 100% analytical sensitivity with a detection limit of Ct = 38.
- Colorimetric LAMP detected 102 plasmid copies with 91.67% sensitivity and no cross-reactivity to other avian viruses.
- Field testing confirmed 100% concordance with real-time PCR across 69 samples.

## Abstract

Avian influenza viruses (AIV) pose a significant threat to poultry health and food security, necessitating rapid and reliable diagnostic methods. This study presents optimized protocols for detecting AIV using Real-Time RT-LAMP and colorimetric LAMP with multiple visual indicators (cresol red, malachite green, calcein). Validation demonstrated that Real-Time LAMP with SYBR Green achieved performance comparable to real-time PCR, with a detection limit corresponding to Ct = 38 and 100% analytical sensitivity (95% CI: 80–100). The colorimetric LAMP format showed a sensitivity limit of 102 plasmid copies (Ct = 32 in RT-PCR) with 91.67% analytical sensitivity (95% CI: 76.1–100). Specificity testing confirmed amplification exclusively in AIV samples (subtypes H1–H12), with no cross-reactivity to other avian viruses or negative controls. Although field monitoring during the study period revealed no natural AIV infections, comprehensive parallel testing with real-time PCR established 100% concordance across all field samples (n = 69), confirming the method’s diagnostic reliability. The optimized LAMP protocols provide a robust, rapid detection system suitable for resource-limited settings, enabling early outbreak identification and enhancing AIV surveillance capabilities without requiring complex instrumentation.

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.