Wang Siyuan, Liu Ning, Zheng Lingyan, Cai Gaozhe, Lin Jianhan
Key Laboratory of Agricultural Information Acquisition Technology, Ministry of Agriculture and Rural Affairs, China Agricultural University, Beijing 100083, China.
Lab Chip. 2020 Jun 30;20(13):2296-2305. doi: 10.1039/d0lc00290a.
Rapid screening of foodborne pathogens is key to prevent food poisoning. In this study, a lab-on-chip device was developed for rapid, automatic and sensitive detection of viable Salmonella typhimurium using loop-mediated isothermal amplification (LAMP) and smartphone real-time turbidity monitoring. First, magnetic nanoparticles (MNPs) coated with anti-Salmonella capture antibodies in propidium monoazide (PMA) were fully mixed with bacterial samples using two active magnetic stirring mixers at reverse rotating directions, and incubated in the serpentine channel with 470 nm blue light exposure, allowing specific formation of magnetic bacteria and sufficient PMA pretreatment of the DNA of dead bacteria. Then, the PMA-treated magnetic bacteria were separated in the separation chamber using the magnetic field and their genomic DNA templates were extracted using lysis buffer at 70 °C. Finally, the viable bacteria's DNA was amplified using LAMP in the detection chamber preloaded with the lyophilized LAMP reagents at 67.5 °C after blocking with paraffin oil to avoid aerosol cross contamination. Finally, the turbidity of the LAMP reaction system was monitored in a real-time manner for the quantitative detection of viable bacteria. The experimental results demonstrated that this device was able to automatically detect viable Salmonella as low as 14 CFU mL-1 in spiked chicken meat supernatants within 1.5 h. This device is very promising to provide a sample-in-result-out solution for the in-field detection of Salmonella and could be easily extended for other foodborne pathogens.
快速筛查食源性病原体是预防食物中毒的关键。在本研究中,开发了一种芯片实验室设备,用于使用环介导等温扩增(LAMP)和智能手机实时浊度监测快速、自动且灵敏地检测鼠伤寒沙门氏菌的活菌。首先,将涂有抗沙门氏菌捕获抗体的磁性纳米颗粒(MNPs)与单叠氮化丙锭(PMA)中的细菌样品在两个反向旋转的主动磁搅拌器作用下充分混合,并在蛇形通道中进行470 nm蓝光照射孵育,使磁性细菌特异性形成,并对死菌的DNA进行充分的PMA预处理。然后,经PMA处理的磁性细菌在分离室中利用磁场进行分离,并用70℃的裂解缓冲液提取其基因组DNA模板。最后,在用石蜡油封闭以避免气溶胶交叉污染后,在预装有冻干LAMP试剂的检测室中于67.5℃下使用LAMP扩增活菌的DNA。最后,实时监测LAMP反应体系浊度以对活菌进行定量检测。实验结果表明,该设备能够在1.5小时内自动检测出加标鸡肉上清液中低至14 CFU mL-1的活菌。该设备非常有前景为沙门氏菌的现场检测提供一种样品进结果出的解决方案,并可轻松扩展用于其他食源性病原体的检测。