A reversible valve-assisted chip coupling with integrated sample treatment and CRISPR/Cas12a for visual detection of Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus (V. parahaemolyticus) is regarded as a major cause of seafood-associated illnesses, which has aroused widespread public concern. Here, a rapid and convenient detection method for V. parahaemolyticus detection was established by a reversible valve-assisted chip coupling with CRISPR/Cas12a. With optimized lysis buffer, loop mediated isothermal amplification (LAMP) reagents and CRISPR reagents, the whole detection process from sampling to results could be finished within 50 min. The structure of chip was simple and the cost was low. By relying on three reversible rotary valves and the rotation direction-dependent Coriolis pseudo force, the flow order of liquid and the direction of liquid flow could be precisely controlled. The LAMP amplicons were specifically and sensitively identified by CRISPR/Cas12a. Positive amplification would produce green fluorescent signal while negative amplification generated no fluorescent signal, which could be clearly distinguished by the naked eye. With 600 μL of samples processed, the limit of detection (LOD) for both pure cultured V. parahaemolyticus or spiked shrimp samples could achieve 30 copies/reaction. These illustrated the established method displayed great feasibility for real samples detection. In the future, the chip could also combine with other amplification reactions, like PCR or recombinase polymerase amplification reaction (RPA), to conduct detection by changing the corresponding lyophilized amplification reagents. Overall, the proposed detection platform displays great potential for food safety analysis and clinical diagnostics, especially in resource-limited areas.