JZUS - Journal of Zhejiang University SCIENCE

Journal of Zhejiang University SCIENCE B 2023 Vol.24 No.8 P.749-754

http://doi.org/10.1631/jzus.B2200705

Rapid visual detection of Vibrio parahaemolyticus by combining LAMP-CRISPR/Cas12b with heat-labile uracil-DNA glycosylase to eliminate carry-over contamination

Author(s): Fang WU, Chen LU, Wenhao HU, Xin GUO, Jiayue CHEN, Zhidan LUO
Affiliation(s): Jiangsu Key Laboratory of Marine Biological Resources and Environment, Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Ocean University, Lianyungang 222005, China; more
Corresponding email(s): lzd@jou.edu.cn
Key Words: UDG, LAMP, CRISPR/Cas12b, Vibrio parahaemolyticus, One-pot detection

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Abstract: vibrio parahaemolyticus is a major pathogen frequently found in seafood. Rapid and accurate detection of this pathogen is important for the control of bacterial foodborne diseases and to ensure food safety. In this study, we established a one-pot system that combines uracil-DNA glycosylase (UDG), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12b (Cas12b) for detecting V. parahaemolyticus in seafood. This detection system can effectively perform identification using a single tube and avoid the risk of carry-over contamination.

结合LAMP-CRISPR/Cas12b与热敏感尿嘧啶DNA糖苷酶实现可消除残留污染的副溶血弧菌快速可视化检测

吴芳^1,2，卢辰^1,2，胡文灏^1,2，郭馨³，陈佳玥^1,2，罗志丹^1,2
¹江苏海洋大学，江苏省海洋生物资源与环境重点实验室，江苏省海洋药物活性分子筛选重点实验室，中国连云港市， 222005
²江苏海洋大学，江苏省海洋生物产业技术协同创新中心，中国连云港市， 222005
³江苏百时美生物科技有限公司，中国连云港市， 222005
摘要: 作为海产品中的主要致病菌，副溶血弧菌的快速准确检测对于海水健康养殖和避免副溶血弧菌相关食源性疾病的发生至关重要。本研究结合尿嘧啶DNA糖苷酶（UDG)、环介导等温扩增（LAMP)和CRISPR/Cas12b技术，建立了海产品中副溶血弧菌的一锅法检测技术。该检测系统实现了一管化检测，并可避免残留污染的风险。我们通过优化dNTP混合物中dTTP/dUTP比例，并筛选出最优sgRNA。该方法在最优条件下，对副溶血弧菌纯培养物的检出限低至1×10² CFU/mL，在虾肉样品的检出限低至1×10² CFU/g。该方法对其他微生物病原体无交叉反应，且与荧光定量PCR结果符合率为100%。

关键词：尿嘧啶糖苷酶；环介导等温扩增；CRISPR/Cas12b；副溶血弧菌；一锅法检测

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结合LAMP-CRISPR/Cas12b与热敏感尿嘧啶DNA糖苷酶实现可消除残留污染的副溶血弧菌快速可视化检测

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