Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay.
Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay.
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DOI：
10.1016/j.bios.2011.09.029
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发表时间：
2011-12
影响因子：
12.6
通讯作者：
P. Zhu;D. Shelton;Shuhong Li;D. Adams;J. Karns;Platte T. Amstutz;Cha-Mei Tang
中科院分区：
文献类型：
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作者：
P. Zhu;D. Shelton;Shuhong Li;D. Adams;J. Karns;Platte T. Amstutz;Cha-Mei Tang
传统的以培养为基础的检测E.大肠杆菌O 157：H7在食品和水源中的检测耗时，结果可能不明确，需要通过生化检测和PCR进一步确认。在培养前进行快速免疫测定以鉴定推定阳性样品将节省大量时间和资源。免疫磁性分离（IMS）技术通常用于分离E。大肠杆菌O 157：H7，通常与培养检测结合，然后进行生化和血清学确认。本研究将免疫磁性荧光技术与荧光免疫分析技术相结合，建立了一种检测大肠杆菌的新方法。大肠杆菌O 157：H7。E. coli O 157：H7细胞首先被抗O 157抗体包被的磁珠捕获，然后被荧光检测抗体识别，形成免疫夹心复合物。随后将该复合物解离，用于用Signalyte™-II分光荧光计测量荧光强度。进行实验以评价测定的线性和灵敏度。当细胞浓度<105 cells/mL时，通过培养平板和定量实时PCR测定，捕获效率大于98%。由于珠结合能力的限制，捕获效率在较高的细胞浓度下降低。在较低的细胞浓度（10- 104 cells/mL）下，Cy 5解离液的荧光强度与E. coli 157：H7细胞浓度。检测限为10 CFU/mL水。由于不需要富集，与通常需要24小时孵育进行预富集，然后进行确证试验的现有技术相比，该测定可在不到3小时内完成。
Conventional culture-based methods for detection of E. coli O157:H7 in foods and water sources are time-consuming, and results can be ambiguous, requiring further confirmation by biochemical testing and PCR. A rapid immunoassay prior to cultivation to identify presumptive positive sample would save considerable time and resources. Immunomagnetic separation (IMS) techniques are routinely used for isolation of E. coli O157:H7 from enriched food and water samples, typically in conjunction with cultural detection followed by biochemical and serological confirmation. In this study, we developed a new method that combines IMS with fluorescence immunoassay, termed immunomagnetic fluorescence assay (IMFA), for the detection of E. coli O157:H7. E. coli O157:H7 cells were first captured by anti-O157 antibody-coated magnetic beads and then recognized by a fluorescent detector antibody, forming an immunosandwich complex. This complex was subsequently dissociated for measurement of fluorescence intensity with Signalyte™-II spectrofluorometer. Experiments were conducted to evaluate both linearity and sensitivity of the assay. Capture efficiencies were greater than 98%, as determined by cultural plating and quantitative real-time PCR, when cell concentrations were <105cells/mL. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (10–104cells/mL), the fluorescence intensity of dissociated Cy5 solution was highly correlated with E. coli 157:H7 cell concentrations. The detection limit was 10CFU per mL of water. The assay can be completed in less than 3h since enrichment is not required, as compared to existing techniques that typically require a 24h incubation for pre-enrichment, followed by confirmatory tests.