A centrifugal microfluidic platform for rapid detection of pathogens based on chitosan nucleic acid extraction and RAA-T7-CRISPR/Cas13a nucleic acid detection

Abstract

This study reported a bacterial detection platform using chitosan electrostatic adsorption for nucleic acid purification, RAA-T7-CRISPR/Cas13a system for nucleic acid quantification and centrifugal microfluidic platform for full-automatic operation. First, the nucleic acids in the lysed bacterial sample were captured by chitosan modified in the microfluidic PDMS channel through electrostatic adsorption, followed by washing with sodium acetate buffer solution. Then, these captured nucleic acid templates were eluted using Tris-HCl buffer solution through restoring the electrical neutrality of chitosan. Finally, the eluted nucleic acid templates were transferred into the RAA-T7-CRISPR/Cas13a system and isothermally amplified at 37 °C, followed by real-time detection of fluorescent products. A centrifugal microfluidic chip was elaborately designed with a chitosan modified channel, eight functional chambers and three siphon valves to achieve sequential capture, washing, elution, amplification and detection of nucleic acids. This platform has the capacity of automatically detecting Salmonella within 1 h. The detection limit of this platform was 10 CFU/mL in chicken supernatant under the use of optimal conditions. The average recovery and standard deviation of this platform are 106.9% and 6.4%, respectively.