μLAMP-LFA: A nucleic acid analysis platform for the field diagnosis of pathogens in aquatic animals.

BACKGROUND

Aquatic animal diseases cause significant economic losses in aquaculture. Effective pathogen diagnostics enable targeted treatment measures. Traditional detection methods primarily rely on polymerase chain reaction. However, due to the bulky and expensive nature of the equipment, on-site testing is impractical. Furthermore, the process requires low-temperature preservation, transportation to centralized laboratories, and analysis by trained personnel. The combination of loop-mediated isothermal amplification (LAMP) and lateral flow assay (LFA) has promoted on-site detection with greater convenience. However, there are still some shortcomings with the current LAMP-LFA technology.

RESULTS

A micro-volume LAMP-LFA platform (μLAMP-LFA) was developed for on-site diagnosis of aquatic animal pathogens. The platform comprised a microfluidic chip and dedicated equipment (mini LAMPer). The μLAMP-LFA chip integrated LAMP and LFA technologies. After rotating the chip, the amplification solution was automatically transferred into the LFA chamber. The hermetically sealed chip effectively prevented false-positive results caused by aerosols. Additionally, the chip included a dedicated compartment for separate storage of dilution solutions, facilitating micro-volume LAMP-LFA. The μLAMP-LFA chip demonstrated excellent flow control, fluidity, and mixing capabilities. The rapid heating rate and stable temperature of the mini LAMPer ensured optimal conditions for LAMP reactions. The platform detected white spot syndrome virus and Salmonella at concentrations as low as 28 copies/μL and 15 copies/μL, respectively. The dual pathogen detection capability of the platform was validated using real shrimp samples.

SIGNIFICANCE: Compared to conventional LAMP-LFA, μLAMP-LFA minimizes reagent usage, drastically lowering detection costs. More importantly, the pre-stored dilution solution in the chip plays a critical role in cooling the amplified products, preventing potential test strip failure. Its simple operation, portability and low cost grant the platform significant potential for field detection of pathogens in aquatic animals. In addition, it can also be applied to food safety, agricultural monitoring, environmental analysis, and other on-site detection applications.