A highly Sensitive ERA-CRISPR/Cas13a Assay for Detection of Vibrio vulnificus.

Abstract

Background: Vibrio vulnificus is a halophilic marine bacterium widely distributed in coastal environments and seafood products and is responsible for severe human infections and foodborne disease outbreaks. Rapid and sensitive detection of V. vulnificus is therefore critical for clinical diagnosis and seafood safety monitoring.

Objective: The objective of this study was to develop and evaluate a rapid, sensitive, and specific nucleic acid detection method for V. vulnificus suitable for application in complex biological and food matrices.

Methods: An ERA-CRISPR/Cas13a-based nucleic acid detection assay was established by coupling enzymatic recombinase amplification (ERA) with Cas13a-mediated collateral cleavage of fluorescent RNA reporters. The analytical performance of the assay was systematically evaluated using V. vulnificus standard plasmids, artificially contaminated human serum samples, and spiked oyster homogenates.

Results: The assay exhibited high specificity for V. vulnificus, with no cross-reactivity observed for other common Vibrio species. The limit of detection (LOD) was 1 × 10-1 copies/μL for standard plasmid templates. In complex matrices, including human serum and oyster samples, the assay achieved an LOD of 1 × 101 CFU/mL. The entire detection process was completed under isothermal conditions within 45 min.

Conclusions: A sensitive and specific ERA-CRISPR/Cas13a-based assay for the detection of V. vulnificus was successfully developed and validated. The method demonstrates strong analytical performance in both biological and seafood matrices.

Highlights: A rapid isothermal ERA-CRISPR/Cas13a assay for V. vulnificus detection was developed. High specificity was achieved without cross-reactivity to other Vibrio species. The assay enables sensitive detection in serum and oyster samples. The method shows potential for routine seafood safety surveillance and marine pathogen monitoring.