Visual Detection of Vibrio parahaemolyticus using Combined CRISPR/Cas12a and Recombinase Polymerase Amplification

Abstract

Objective: To establish an ultra-sensitive, ultra-fast, visible detection method for Vibrio parahaemolyticus (VP) .

Methods: We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) combined with recombinase polymerase amplification and visual detection (CRISPR/Cas12a-VD).

Results: CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10 ^-18 M (single molecule detection) within 30 min without cross-reactivity against other bacteria. When detecting pure cultures of VP, the consistency of results reached 100% compared with real-time PCR. The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10 ² CFU/g.

Conclusion: The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods, such as real-time PCR, and has great potential for preventing the spread of pathogens.

Keywords: CRISPR/Cas12a-VD; Cross-reactivity; Isothermal amplification; Recombinase polymerase amplification; Vibrio parahaemolyticus; Visual detection.