Molecular Biology

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.

During swarming, high density flagella-driven bacteria migrate collectively in a swirling pattern on wet agar surfaces, immersed in a thin viscous fluid layer called “swarm fluid”. Though the fluid environment has essential role in the emergence of swarming behavior, the microscopic mechanisms of it in mediating the cooperation of bacteria populations are not fully understood. Here, instead of micro-sized tracers used in previous research, we use gold nanorods as single particle tracers to probe the dynamics of the swarm fluid. This protocol includes five major parts: (1) the culture of swarming bacterial colony; (2) the preparations of gold nanorod tracers and the micro-spraying technique which are used to put the nanotracers into the upper fluid of bacterial swarms; (3) imaging and tracking; (4) other necessary control experiments; (5) data analysis and fitting of physical models. With this method, the nano-sized tracers could move long distances above motile cells without direct collisions with the bacteria bodies. In this way, the microscopic dynamics of the swarm fluid could be tracked with high spatiotemporal resolution. Moreover, the comprehensive analysis of multi-particle trajectories provides systematic visualization of the fluid dynamics. The method is promising to probe the fluid dynamics of other natural or artificial active matter systems.

Paper nanobiosensors have been established as an excellent platform for analysis of veterinary and human pathogens causing various diseases. Especially, lateral flow assays or biosensors ideal for sensitive, rapid, robust and accurate analysis in laboratory setups and on-site analysis. Viral RNA detection is of great importance for public health as well as animal health protection. In that aspect, the present protocol focuses on the development of functionalized gold nanoparticle-based lateral flow biosensor for fish nervous necrosis virus (Nodavirus) nucleic acids detection. Total viral RNA, isolated from fish samples was subjected to reverse transcription PCR amplification and the amplification products were mixed with specific oligonucleotide probe. A red test line was formed when nodavirus product was present. The proposed assay has great implications on basic research since it eliminates the need for time-consuming, cumbersome electrophoresis protocols and could be adjusted for use on the site of fish culture by fish farmers. Disease monitoring by such bioanalytical platforms without time consuming and costly procedures would have great impact on the aquaculture and environmental safety.