A Novel Real-Time Loop-Mediated Isothermal Amplification Combined with Immunomagnetic Beads Separation and Ethidium Bromide Monoazide Treatment for Rapid and Ultrasensitive Detection of Viable Escherichia coli O157:H7 in Milk
A Novel Real-Time Loop-Mediated Isothermal Amplification Combined with Immunomagnetic Beads Separation and Ethidium Bromide Monoazide Treatment for Rapid and Ultrasensitive Detection of Viable Escherichia coli O157:H7 in Milk
复制标题
新型实时环介导等温扩增结合免疫磁珠分离和溴化乙锭单叠氮化物处理，可快速、超灵敏地检测牛奶中活大肠杆菌 O157:H7
DOI：
10.1007/s12161-020-01932-y
复制
发表时间：
2021-01-06
影响因子：
2.9
通讯作者：
Wu, Guo-Ping
中科院分区：
文献类型：
--
作者：
Chen, Hu;Li, Ya-Ke;Wu, Guo-Ping
In this study, a real-time loop-mediated isothermal amplification (Rti-LAMP) combined with immunomagnetic beads separation (IMS) and ethidium bromide monoazide (EMA) treatment was developed for the rapid and ultrasensitive detection of viable Escherichia coli (E. coli) O157:H7 in milk without enrichment. Polyclonal antibodies against E. coli O157:H7 flagellum and specific immunomagnetic beads (IMBs) were prepared. The capture efficiency of target bacteria was more than 95% by adding 50 mu L of prepared IMBs into 1 mL of E. coli sample for incubation at 37 degrees C for 20 min. Then, Gly-HCl buffer (0.2 M, pH 2.2) was a key to the effective eluting and separating of bacteria from IMB-bacterial complexes. The eluted bacteria were resuspended with 20 mu L TZ lysing solution and boiled at 99.5 degrees C for 10 min, as a DNA template of Rti-LAMP. The development of IMS-Rti-LAMP assay could specifically detect viable E. coli O157:H7 as low as 2.7 CFU/mL in pure culture. Furthermore, the optimum volume for detection of E. coli O157:H7 in milk was 1 mL of the sample by IMS-Rti-LAMP assay. Before Gly-HCl elution, 4 mu g/mL of EMA was used to treat bacterial cells which significantly inhibited DNA amplification derived from dead cells in Rti-LAMP. The limit of detection (LOD) of viable E. coli O157:H7 in milk was 6 CFU/mL by the IMS-EMA-Rti-LAMP, and the whole assay could be completed in 4 h. The development of IMS-EMA-Rti-LAMP assay was a rapid, ultrasensitive, and specific detection of viable E. coli O157:H7 in milk without enrichment.
