Rapid and Sensitive Detection of Vibrio vulnificus Using CRISPR/Cas12a Combined With a Recombinase-Aided Amplification Assay

Abstract

<i>Vibrio vulnificus</i> is an important zoonotic and aquatic pathogen and can cause vibriosis in humans and aquatic animals (especially farmed fish and shrimp species). Rapid and sensitive detection methods for <i>V. vulnificus</i> are still required to diagnose human vibriosis early and reduce aquaculture losses. Herein, we developed a rapid and sensitive diagnostic method comprising a recombinase-aided amplification (RAA) assay and the CRISPR/Cas12a system (named RAA-CRISPR/Cas12a) to detect <i>V. vulnificus</i>. The RAA-CRISPR/Cas12a method allows rapid and sensitive detection of <i>V. vulnificus</i> in 40 min without a sophisticated instrument, and the limit of detection is two copies of <i>V. vulnificus</i> genomic DNA per reaction. Meanwhile, the method shows satisfactory specificity toward non-target bacteria and high accuracy in the spiked blood, stool, and shrimp samples. Therefore, our proposed rapid and sensitive <i>V. vulnificus</i> detection method, RAA-CRISPR/Cas12a, has great potential for early diagnosis of human vibriosis and on-site <i>V. vulnificus</i> detection in aquaculture and food safety control.