A loop-mediated isothermal amplification-based microfluidic chip for triplex detection of shrimp pathogens.

Area of Science:

• Aquaculture
• Pathogen detection
• Microfluidics

Background:

• Decapod iridescent virus 1 (DIV1), White spot syndrome virus (WSSV), and Enterocytozoon hepatopenaei (EHP) are significant threats to shrimp aquaculture.
• Early and accurate pathogen detection is crucial for disease management and preventing widespread outbreaks.

Purpose of the Study:

• To develop a novel, rapid, and simultaneous detection system for DIV1, WSSV, and EHP.
• To create a microfluidic chip-based assay utilizing loop-mediated isothermal amplification (LAMP) for field-deployable diagnostics.

Main Methods:

• Development of a triplex loop-mediated isothermal amplification (LAMP) assay integrated into a microfluidic chip.
• Optimization of reaction conditions for simultaneous detection of three shrimp pathogens.
• Validation of the assay using clinical samples from Penaeus vannamei and comparison with quantitative polymerase chain reaction (qPCR).

Main Results:

• The microfluidic chip achieved limits of detection of 10 copies/reaction for EHP and DIV1, and 10^2 copies/reaction for WSSV.
• The entire detection process was completed in 40 minutes at 63°C with 100% specificity.
• The assay demonstrated excellent stability, reproducibility, and comparable performance to qPCR when tested on 94 clinical samples.

Conclusions:

• The developed LAMP-based microfluidic chip enables rapid, simultaneous, and accurate detection of three major shrimp pathogens.
• This technology offers a valuable tool for field diagnostics and routine monitoring in aquaculture, aiding in disease control and prevention.