小型化Spreeta SPR免疫传感器在大肠杆菌O157：H7检测中的研究

发布时间：2019-01-19 21:06

【摘要】：大肠杆菌0157:H7(E. coli 0157:H7)作为危害最大的食源性致病菌之一,在我国存在流行暴发的可能性和感染的严重性。由于传统检测方法存在操作繁琐、费力、耗时等缺点,因此急需研制一种快速准确的检测方法。本课题调查了目前国际上诸多新型检测技术,包括生物传感器。根据Spreeta SPR传感器的优势,最终将Spreeta SPR传感器与细胞裂解法作为主要研究手段与内容,实现对大肠杆菌O157：H7的检测,并对其进行了较为深入的探讨和研究。本课题的主要研究内容和研究结果如下： (1)用不同浓度葡萄糖溶液评估Spreeta SPR传感器性能,葡萄糖浓度与折射率间的相关系数接近1,用去离子水冲洗后回复基线,得出该传感器性能良好。 (2)通过亲和素及生物素标记抗体的响应结果的重复性实验可知该传感器重复性高,为Spreeta SPR免疫传感器用于检测大肠杆菌O157：H7提供了理论基础,其检测限为3×106 cfu mL-1,基本满足检测需求。 (3)比较BIACORE SPR免疫传感器,用直接法检测大肠杆菌O157：H7的检测限为3×105 cfu mL-1,检测灵敏度与酶联免疫吸附法(ELISA)的灵敏度相当,Spreeta SPR免疫传感器的灵敏度有待提高。 (4)研究选用细胞裂解技术与Spreeta SPR免疫传感器结合来降低检测限,样品菌液超声处理后,检测限下降为3×104 cfu mL-1,相关系数为0.98。结果表明细胞裂解技术能有效降低传感器的检测限。 (5)采用大肠杆菌DH 5α和李斯特菌作为验证菌检验传感器对大肠杆菌0157：H7的特异性,得出传感器对目标菌的特异性良好,对验证菌的吸附很小。
[Abstract]:E. coli 0157:H7 (E. coli 0157:H7, as one of the most harmful foodborne pathogens, has the possibility of epidemic outbreak and the severity of infection in China. Because of the disadvantages of complicated operation, laborious operation and time consuming in the traditional detection methods, it is urgent to develop a fast and accurate detection method. In this paper, many new detection techniques, including biosensors, have been investigated. According to the advantage of Spreeta SPR sensor, the Spreeta SPR sensor and cell lysis method were taken as the main research means and content, the detection of O157:H7 in E. coli was realized, and it was discussed and studied deeply. The main research contents and results are as follows: (1) the performance of Spreeta SPR sensor is evaluated with different concentrations of glucose solution. The correlation coefficient between glucose concentration and refractive index is close to 1, and the baseline is restored after rinsing with deionized water. The sensor has good performance. (2) the repeatability of the response results of avidin and biotinylated antibody showed that the sensor had high reproducibility, which provided a theoretical basis for the detection of Escherichia coli O157:H7 by Spreeta SPR immunosensor. The detection limit was 3 脳 10 ~ 6 cfu mL-1,. Basically meet the test needs. (3) comparing the sensitivity of BIACORE SPR immunosensor, the detection limit of direct detection of Escherichia coli O157:H7 is 3 脳 10 ~ 5 cfu mL-1,. The sensitivity of, Spreeta SPR immunosensor is comparable to that of (ELISA) by enzyme linked immunosorbent assay (Elisa). The sensitivity of, Spreeta SPR immunosensor needs to be improved. (4) the detection limit was decreased to 3 脳 10 ~ 4 cfu mL-1, after ultrasonic treatment with Spreeta SPR immunosensor and cell lysis technique. The correlation coefficient was 0.98. The results show that the cell lysis technique can effectively reduce the detection limit of the sensor. (5) Escherichia coli DH 5 伪 and Listeria were used to test the specificity of the biosensor to Escherichia coli 0157:H7. The results showed that the specificity of the biosensor to the target bacteria was good, and the adsorption of the validation bacteria was very small.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R155.5

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