摘要
      快速检测食品微生物的抗生素抗性基因（ARGs）对于防止ARGs通过食物链传播至关重要。本研究开发了基于滑片的免疫磁分离（IMS）结合环介导的等温扩增（LAMP），用于巴氏灭菌乳中具有四环素抗性基因tetL的蜡样芽孢杆菌的快速、特异、灵敏和多样本检测。首先，涂有抗B抗体的磁性纳米颗粒。将蜡样多克隆抗体与样品在滑片的磁性捕获室中混合，孵育1h后，蜡样芽孢杆菌（105CFU/mL）的捕获效率可达80%。然后，使用加热块将捕获的细菌在裂解室中煮沸，并将提取的DNA作为模板加入LAMP反应中。最后，在65°C下对蜡样芽孢杆菌tetL基因进行了50分钟的实时荧光LAMP检测。在最佳条件下，IMS-LAMP可以在纯培养中检测到11.6CFU/mL的具有tetL的蜡样芽孢菌。此外，基于滑动芯片的IMS-LAMP在2小时内检测巴氏灭菌乳中具有tetL基因的蜡样芽孢杆菌的极限为21.5 CFU/mL。因此，该方法有可能用于快速检测巴氏灭菌奶中具有四环素抗性基因tetL的蜡样芽胞杆菌，并可扩展到其他抗性基因。
Abstract
Rapid detection of antibiotic resistance genes (ARGs) of food microorganisms is critical for preventing the spread of ARGs via the food chain. In this study, slipchip-based immunomagnetic separation (IMS) combined with loop-mediated isothermal amplification (LAMP) was developed for rapid, specific, sensitive and multi-sample detection of Bacillus cereus with tetracycline resistance gene tetL in pasteurized milk. First, the magnetic nanoparticles coated with anti-B. cereus polyclonal antibodies were mixed with samples in the magnetic capture chamber of the slipchip, and the capture efficiency of Bacillus cereus (105 CFU/mL) can reach 80% after incubation for 1h. Then, the captured bacteria were boiled in the lysis chamber using a heating block and the extracted DNA was added into the LAMP reaction as templates. Finally, real-time fluorescent LAMP detection for tetL gene of Bacillus cereus was performed at 65 °C for 50 min. Under optimal conditions, the IMS-LAMP could detect 11.6 CFU/mL of Bacillus cereus with tetL gene in pure culture. Furthermore, the limit of detection of Bacillus cereus with tetL gene in pasteurized milk was 21.5 CFU/mL by the slipchip-based IMS-LAMP within 2 h. Therefore, this method is potentially useful for the rapid detection of Bacillus cereus with tetracycline resistance gene tetL in pasteurized milk and could be extended for other resistance genes.

https://www.sciencedirect.com/science/article/abs/pii/S0956713522003152