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%.
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Acknowledgments
This work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions of China, the Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX2021-038), and the Lianyungang Science and Technology Program (No. CG2232). We thank Mr. Xiang ZHOU (Lianyungang Institute of Food and Drug Control, Lianyungang, China) and Dr. Juan LI (Wuhan Institute for Food and Cosmetic Control, Wuhan, China) for kindly providing the samples used in this study. We also thank Dr. Yong XUE (Jiangsu Ocean University, Lianyungang, China) for his critical review of this manuscript.
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Zhidan LUO conceived and designed the experiments. Fang WU, Wenhao HU, Xin GUO, and Jiayue CHEN performed the experimental research. Fang WU and Chen LU analyzed the data. Zhidan LUO and Chen LU wrote this manuscript. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Fang WU, Chen LU, Wenhao HU, Xin GUO, Jiayue CHEN, and Zhidan LUO declare that they have no conflict of interest.
All institutional and national guidelines for the care and use of laboratory animals were followed.
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Detailed methods are provided in the electronic supplementary materials of this paper.
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Materials and methods; Table S1; Fig. S1
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Wu, F., Lu, C., Hu, W. et al. Rapid visual detection of Vibrio parahaemolyticus by combining LAMP-CRISPR/Cas12b with heat-labile uracil-DNA glycosylase to eliminate carry-over contamination. J. Zhejiang Univ. Sci. B 24, 749–754 (2023). https://doi.org/10.1631/jzus.B2200705
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DOI: https://doi.org/10.1631/jzus.B2200705