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CE–RAA–CRISPR Assay: A Rapid and Sensitive Method for Detecting Vibrio parahaemolyticus in Seafood

Authors :
Xinrui Lv
Weiwei Cao
Huang Zhang
Yilin Zhang
Lei Shi
Lei Ye
Source :
Foods, Vol 11, Iss 12, p 1681 (2022)
Publication Year :
2022
Publisher :
MDPI AG, 2022.

Abstract

Vibrio parahaemolyticus is one of the major pathogenic Vibrio species that contaminate seafood. Rapid and accurate detection is crucial for avoiding foodborne diseases caused by pathogens and is important for food safety management and mariculture. In this study, we established a system that combines chemically enhanced clustered regularly interspaced short palindromic repeats (CRISPR) and recombinase-aided amplification (RAA) (CE–RAA–CRISPR) for detecting V. parahaemolyticus in seafood. The method combines RAA with CRISPR-associated protein 12a (Cas12a) for rapid detection in a one-pot reaction, effectively reducing the risk of aerosol contamination during DNA amplifier transfer. We optimized the primers for V. parahaemolyticus, determined the optimal crRNA/Cas12a ratio, and demonstrated that chemical additives (bovine serum albumin and L-proline) could enhance the detection capacity of Cas12a. The limit of detection (at optimal conditions) was as low as 6.7 × 101 CFU/mL in pure cultures and 7.3 × 101 CFU/g in shrimp. Moreover, this method exhibited no cross-reactivity with other microbial pathogens. The CE–RAA–CRISPR assay was compared with the quantitative polymerase chain reaction assay using actual food samples, and it showed 100% diagnostic agreement.

Details

Language :
English
ISSN :
23048158
Volume :
11
Issue :
12
Database :
Directory of Open Access Journals
Journal :
Foods
Publication Type :
Academic Journal
Accession number :
edsdoj.39a784af28d949dca50c9f2f5cdb34ec
Document Type :
article
Full Text :
https://doi.org/10.3390/foods11121681