Rapid isolation and detection of aquaculture pathogens in an integrated microfluidic system using loop-mediated isothermal amplification

Abstract

Aquaculture is an economically important activity for maritime countries. However, the spread of infectious diseases among aquaculture species have a serious impact on this industry. Therefore, the early detection of pathogens is crucial for protecting aquaculture species with a premium commercial value. This study reports an integrated microfluidic system for the rapid detection of aquaculture pathogens. In this new device, the DNA (deoxyribonucleic acid) of the target pathogen was first isolated by magnetic beads coated with specific nucleotide probes after cell lysis. Then the extracted DNA fragments were amplified by the loop-mediated isothermal amplification (LAMP) process and the amplified products were detected optically. This device contained normally closed microvalves, micropumps, LAMP reaction chambers and wash units such that the entire process for isolation of pathogen DNA, nucleic acid amplification and optical detection was performed automatically in the developed microfluidic system. Experimental data showed that the developed system can automatically complete the entire process within 65min with a detection limit of 20 copies. Another advantage of this device is that the chip is comprised of four identical modules. Therefore, this device could carry out detection of four different pathogens simultaneously. In this study, four pathogens common to cultured ornamental fishes, namely Streptococcus agalactiae, koi herpes virus, Iridovirus and Aeromonas hydrophila were analyzed by this integrated device. This device successfully completed four simultaneous detections from infected fishes in a short period of time. Compared to previous studies, this microfluidic system could complete all processes involved in pathogen detection including sample pre-treatment, nucleic acid amplification and optical detection without any manual operation. As a whole, the developed microfluidic system is promising for rapid isolation and detection of aquaculture pathogens.