Rapid and visual detection of Listeria monocytogenes by combining one-pot LAMP–CRISPR/Cas12b with lateral flow assay

IF 5.4 1区农林科学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Food microbiology Pub Date : 2026-04-01 Epub Date: 2025-11-04 DOI:10.1016/j.fm.2025.104977

Xuelan Liu , Yuting Zheng , Zhiwei Chen , Shiqi Wang , Hongyan Liao , Jianye Jia , Guijun Wang , Jialing Wang , Chunyan Yuan , Xiaoxi Guo , Yuelan Yin , Qinghai Hu

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引用次数: 0

Abstract

Listeria monocytogenes, the leading cause of fatalities worldwide among foodborne pathogens, poses serious risks to food safety and public health. Therefore, a rapid and accurate detection method is crucial for early interception and effective management. In this study, a one-pot LAMP–CRISPR/Cas12b detection system based on the lmo0753 gene was developed for rapid detection of L. monocytogenes by combining loop-mediated isothermal amplification (LAMP) with a CRISPR/Cas12b assay. Further integration of a lateral flow assay (LFA) to develop a LAMP–CRISPR/Cas12b–LFA assay enabled direct detection of the results on the strips with the naked eye. Nine L. monocytogenes strains belonging to eight serotypes tested positive with both the one-pot LAMP–CRISPR/Cas12b and LAMP–CRISPR/Cas12b–LFA assays. Two assays did not show cross-reactivity with L. innocua and eight other foodborne bacteria. The limits of detection were 10 CFU/mL for pure culture and 20 CFU/g for spiked pork samples. Moreover, the enrichment time was substantially shortened to 3 h for pork samples spiked with only L. monocytogenes F2365, and 4–5 h for pork samples spiked with mixed bacteria. In addition, with one-pot LAMP-CRISPR/Cas12b detection, 5 of 66 fresh pork samples, 1 of 20 ready-to-eat food samples, and 2 of 24 raw milk samples tested positive for L. monocytogenes, in agreement with the results obtained through a culture based standard method. Thus, this study established one-pot LAMP–CRISPR/Cas12b and LAMP–CRISPR/Cas12b–LFA assays for rapid, visual detection of L. monocytogenes in food samples.

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LAMP-CRISPR /Cas12b单锅法与横向流动法联合检测单核增生李斯特菌

单核细胞增生李斯特菌是全世界食源性病原体中导致死亡的主要原因，对食品安全和公共卫生构成严重风险。因此，快速准确的检测方法对于早期拦截和有效管理至关重要。本研究基于lmo0753基因，将环介导等温扩增（loop-mediated isothermal amplification， LAMP）技术与CRISPR/Cas12b技术相结合，建立了单锅LAMP - CRISPR/Cas12b检测系统，用于单核增生乳杆菌的快速检测。进一步整合横向流动试验（LFA）以开发LAMP-CRISPR / Cas12b-LFA试验，从而可以用肉眼直接检测试纸上的结果。LAMP-CRISPR /Cas12b和LAMP-CRISPR /Cas12b - lfa对8种血清型的9株单增乳杆菌均呈阳性。两项试验未显示与innocul和其他8种食源性细菌的交叉反应。纯培养的检出限为10 CFU/mL，加标猪肉样品的检出限为20 CFU/g。此外，仅添加单核增生乳杆菌F2365的猪肉样品的富集时间大幅缩短至3 h，混合细菌添加的猪肉样品的富集时间为4-5 h。此外，通过一锅LAMP-CRISPR/Cas12b检测，66份新鲜猪肉样品中有5份、20份即食食品样品中有1份、24份原料奶样品中有2份单核细胞增生乳杆菌阳性，与基于培养的标准方法检测结果一致。因此，本研究建立了单锅LAMP-CRISPR /Cas12b和LAMP-CRISPR /Cas12b - lfa检测食品样品中单核细胞增生乳杆菌的方法。

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来源期刊

Food microbiology 工程技术-生物工程与应用微生物

CiteScore

11.30

自引率

3.80%

发文量

179

审稿时长

44 days

期刊介绍： Food Microbiology publishes original research articles, short communications, review papers, letters, news items and book reviews dealing with all aspects of the microbiology of foods. The editors aim to publish manuscripts of the highest quality which are both relevant and applicable to the broad field covered by the journal. Studies must be novel, have a clear connection to food microbiology, and be of general interest to the international community of food microbiologists. The editors make every effort to ensure rapid and fair reviews, resulting in timely publication of accepted manuscripts.