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Detection of Vibrio parahaemolyticus by one-pot LAMP-CRISPR/Cas12b combined with immunomagnetic beads

文献类型： 外文期刊

作者： Wang, Shiqi 1 ; Gao, Yongheng 1 ; Miao, Yiqin 1 ; Jiang, Wei 1 ; Zhang, Xingyu 1 ; Zheng, Yuting 1 ; Wang, Jialing 1 ; Zhang, Xiaoying 1 ; Liao, Hongyan 1 ; Li, Yunsheng 2 ; Liu, Ya 2 ; Hu, Qinghai 1 ;

作者机构： 1.Chinese Acad Agr Sci, Shanghai Vet Res Inst, 518 Ziyue Rd, Shanghai 200241, Peoples R China

2.Anhui Agr Univ, Coll Anim Sci & Technol, 130 West Changjiang Rd, Hefei 230036, Peoples R China

关键词： Vibrio parahaemolyticus; Loop-mediated isothermal amplification; (LAMP); LAMP-CRISPR/cas12b; Lateral flow assay; Immunomagnetic beads; Rapid detection

期刊名称：FOOD CONTROL （ 影响因子：6.3； 五年影响因子：6.1 ）

ISSN： 0956-7135

年卷期： 2025 年 172 卷

页码：

收录情况： SCI

摘要： In this study, loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12b (Cas12b) were combined to establish a one-pot LAMPCRISPR/Cas12b method for the rapid detection of total Vibrio parahaemolyticus based on the opaR gene. The detection results can be observed using a fluorescence signal detector, or be visualized by naked eye through integration with a lateral flow assay (LAMP-CRISPR/Cas12b-LFA). This method could specifically distinguish V. parahaemolyticus from three other Vibrio species and eight common foodborne bacteria. The detection limits of LAMP-CRISPR/Cas12b and LAMP-CRISPR/Cas12b-LFA were 3.7 x 101 colony forming units (CFU)/mL in pure cultures, and 3.7 x 101 CFU/g in bass meat spiked with mixed bacteria without enrichment. Combined with V. parahaemolyticus-specific monoclonal antibodies-labelled immunomagnetic beads (IMB) during sample pretreatment, the enrichment time for detection could be shortened to 1 h for pure culture V. parahaemolyticus (2 CFU/mL), and 2 h for mixed bacteria (V. parahaemolyticus, Salmonella Typhimurium, Escherichia coli, and Staphylococcus aureus; 2 CFU/mL of each species) supplemented with bass meat. Thus, the total time for one-pot LAMP-CRISPR/Cas12b assay, including sample enrichment, IMB separation, DNA extraction, one-pot testing and LFA assay, was less than 4 h. In addition, six out of 50 gill samples of marine fish were positive for V. parahaemolyticus by IMB-LAMP-CRISPR/Cas12b testing, which was completely consistent with the results obtained using a standard method based on bacterial culture. Therefore, a rapid, sensitive, and visual method for detection of V. parahaemolyticus was developed.

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