A real-time isothermal amplification based portable microfluidic system for simple and reliable detection of Vibrio splendidus

Abstract

The spread of infectious diseases among aquaculture species has a serious impact on the aquaculture industry. Simple, specific and low-cost detection methods are urgently needed for early diagnosis and timely treatment, particularly for on-site identifying and tracking of pathogens. Vibrio splendidus (V. splendidus) is regarded as one of the main pathogenic bacteria causing skin ulcerative syndrome in cultured sea cucumbers, leading to massive mortality and severe economic losses. We herein present a microfluidic-based real-time fluorogenic loop-mediated isothermal amplification (LAMP) system for simple and reliable detection of V. splendidus. A LAMP primer set with six primers (arsB1) specifically targeting the arsB gene of V. splendidus was successfully designed and tested on the portable microfluidic system for the first time. Only a single step of sample loading using a pipette is required to fill an array of reaction wells (with 10 or 18 wells) in a disposable chip for multiplex detection. A dedicated plastic shell is then utilized to tightly seal the openings of the chip by buckling to prevent contamination and evaporation. Up to four chips (one sample per chip) can be held in the stand-alone and inexpensive microdevice simultaneously, enabling on-demand detection of multiple samples in a single run. Reproducible (relatively low intra- and inter-chip variability) and sensitive (as few as ∼20 CFU, Colony-Forming Units, per reaction well) on-chip arsB1-LAMP assay was demonstrated by using diluted lysate of V. splendidus. A linear standard curve (R² > 0.98) was attained over the template concentration range of 5 × 10³ to 5 × 10⁶ CFU mL⁻¹. V. splendidus can be detected in samples containing different bacteria, indicating the feasibility of the portable microfluidic LAMP system for parallel detection of multiple bacterial pathogens. The proposed on-chip LAMP assay is simple to operate, reliable for amplification, flexible in detection and cost-effective in instrumentation and testing, holding great potential for on-site rapid detection and routine monitoring of aquaculture pathogens.