Visual Nanoprobe-Enhanced Loop-Mediated Isothermal Amplification Protocol for Rapid Detection of Infectious Laryngotracheitis Virus from Avian Respiratory Swabs

Abstract

A prompt and accurate diagnosis of respiratory viral diseases in intensive poultry production is essential to safeguard animal health and ensure the economic sustainability of farms. Currently, much effort is being devoted to preventing the spread of the avian influenza virus in farms. However, the diagnosis of other relevant respiratory viruses, as infectious laryngotracheitis virus (ILTV), is also crucial. Indeed, infection by ILTV does lead to substantial economic losses due to high morbidity, reduced growth, and decreased productivity, making rapid detection a critical aspect of disease control. Conventional diagnostics, including PCR and qPCR, while sensitive and specific, require expensive laboratory infrastructure and well-trained personnel, limiting their deployment in field settings where immediate intervention is most valuable. To address these limitations, this protocol describes a portable molecular diagnostic workflow based on loop-mediated isothermal amplification (LAMP) combined with gold nanoparticle-DNA nanoprobes for specific and visual detection of ILTV directly at the point of need. Gold nanoparticles synthesized via the Turkevich method are functionalized with thiolated DNA probes, which undergo full-length, sequence-specific hybridization to LAMP amplicons, enabling a naked-eye colorimetric readout. The procedure integrates streamlined steps for DNA probe preparation, nanoparticle synthesis and assembly, and minimal sample processing, compatible with on-farm deployment. Results obtained with this workflow on field samples demonstrated 100% sensitivity and specificity, matching the performance of gold-standard assays. This approach offers a rapid, cost-effective, and equipment-free detection system of viral pathogens, enabling timely decision-making for disease containment and biosecurity. By overcoming the barriers of conventional diagnostics, this protocol enables producers with powerful tools for efficient monitoring and response to respiratory outbreaks in poultry farms. Key features • Direct ILTV detection in respiratory swabs in 35-45 min, bypassing DNA extraction with a rapid viral lysis step. • Specific colorimetric readout via DNA nanoprobes with visual interpretation, requiring no specialized equipment or lab infrastructure. • Achieves 100% sensitivity and specificity compared to qPCR, with a detection limit of 200 viral copies per reaction; validated in lab conditions with field samples. • Modular design: Enables multiplex and customizable detection of other poultry pathogens, supporting rapid kit development and broad field application.