Abstract:
Objective To establish a rapid detection and typing assay for the detection of Vibrio parahaemolyticus by combining recombinase aided amplification（RAA）with clustered regularly interspaced short palindromic repeats（CRISPR）system.
Methods By purifying CRISPR associated protein Cas12a, RAA primers, crRNA and single-stranded DNA reporter were designed and synthesized, and fluorescence and lateral flow dipstick methods were established to detect V. parahaemolyticus. The bacterial nucleic acid was extracted by boiling method, and the product amplified by RAA was added to the CRISPR-Cas12a detection system to test the sensitivity and specificity of the assay.
Results In this study, 110 strains of V. parahaemolyticus and 121 strains of other intestinal diarrheagenic bacteria were detected, and the coincidence rate was 99.1%. The minimum detection limit of bacteria was 103 CFU/mL, and the minimum detection limit of nucleic acid concentration of CRISPR-Cas12a was 77.5 pmol/L. The sensitivity of CRISPR-Cas12a was higher than that of agarose gel electrophoresis. For 50 strains of V. parahaemolyticus with tdh and 12 strains of V. parahaemolyticus without tdh, one strain of V. parahaemolyticus with tdh was not identified, the successful identification rate was 98.4%.
Conclusion CRISPR-RAA can be used for the rapid detection of V. parahaemolyticus and tdh gene with high specificity and sensitivity.
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