Rotary Valve-Assisted Fluidic System Coupling with CRISPR/Cas12a for Fully Integrated Nucleic Acid Detection.

Abstract

Of late, many nucleic acid analysis platforms have been established, but there is still room for constructing integrated nucleic acid detection systems with high nucleic acid extraction efficiency, low detection cost, and convenient operation. In this work, a simple rotary valve-assisted fluidic chip coupling with CRISPR/Cas12a was established to achieve fully integrated nucleic acid detection. All of the detection reagents were prestored on the fluidic chip. With the aid of the rotary valve and syringe, the liquid flow and stirring can be precisely controlled. The nucleic acid extraction, loop-mediated isothermal amplification (LAMP) reaction, and CRISPR detection could be completed in 80 min. A clean reservoir and an air reservoir on the fluidic chip were designed to effectively remove the remaining ethanol. With Vibrio parahaemolyticus as the targets, the detection sensitivity of the fluidic chip could reach 3.1 × 101 copies of target DNA per reaction. A positive sample could be sensitively detected by CRISPR/Cas12a to produce a green fluorescent signal, while a negative sample generated no fluorescent signal. Further, the fluidic chip was successfully applied for detection of spiked shrimp samples, which showed the same detection sensitivity. A great feasibility for real-sample detection was showed by the fluidic chip. The proposed detection platform did not need expensive centrifugal instruments or pumps, which displayed its potential to become a powerful tool for food safety analysis and clinical diagnostics, especially in the resource-limited areas.