小型化Spreeta SPR免疫传感器在大肠杆菌O157：H7检测中的研究
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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。
Escherichia coli O157:H7 is one of highly harmful foodborne disease-caused bacteria for the probability of epidemic outbreak and the danger of infection. It is in dire need of simple, accurate and rapid methods for their detection, since the traditional culture method is time consuming and labor-intensive. Various kinds of new detection methods were presented in details in this thesis, according to the the advantages of Spreeta SPR biosensor, the combination of Spreeta SPR biosensor and cell disruption technique was chosen for studies.
The main contents and results of this thesis were as follows:
(1) Different concentrations of glucose were used to assess the Spreeta SPR biosensor, the linear correlation coefficient (R) between refractive index and series of glucose concentrations was close to 1, which showed that the biosensor performance was quite well.
(2) Following the RI response of the streptavidin and biotin-labeled antibody immobilization, it showed us the high repeatability of the Spreeta SPR biosensor, and the limit of detection (LOD) was 3×106 cfu mL-1 for detection of E. coli O157:H7.
(3) The limit of detection for E. coli 0157:H7 was 3×105 cfu mL-1 based on BIACORE SPR immunosensor, as the same of that based on ELISA, it showed that the sensitivity was needed to be improved.
(4) Cell disruption technique was used in this study, the limit of detection has been improved to 3×104 cfu mL-1 after cell disruption, with R2 of 0.98, which showed that Cell disruption technique leaded to a lower LOD.
(5) E. coli DH 5a and Listeria were used to study the specificity of the SPR biosensor, and the resultsshowed that the SPR biosensor had high combination with on the target bacteria, and very low non-target adsorption.
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。
Escherichia coli O157:H7 is one of highly harmful foodborne disease-caused bacteria for the probability of epidemic outbreak and the danger of infection. It is in dire need of simple, accurate and rapid methods for their detection, since the traditional culture method is time consuming and labor-intensive. Various kinds of new detection methods were presented in details in this thesis, according to the the advantages of Spreeta SPR biosensor, the combination of Spreeta SPR biosensor and cell disruption technique was chosen for studies.
The main contents and results of this thesis were as follows:
(1) Different concentrations of glucose were used to assess the Spreeta SPR biosensor, the linear correlation coefficient (R) between refractive index and series of glucose concentrations was close to 1, which showed that the biosensor performance was quite well.
(2) Following the RI response of the streptavidin and biotin-labeled antibody immobilization, it showed us the high repeatability of the Spreeta SPR biosensor, and the limit of detection (LOD) was 3×106 cfu mL-1 for detection of E. coli O157:H7.
(3) The limit of detection for E. coli 0157:H7 was 3×105 cfu mL-1 based on BIACORE SPR immunosensor, as the same of that based on ELISA, it showed that the sensitivity was needed to be improved.
(4) Cell disruption technique was used in this study, the limit of detection has been improved to 3×104 cfu mL-1 after cell disruption, with R2 of 0.98, which showed that Cell disruption technique leaded to a lower LOD.
(5) E. coli DH 5a and Listeria were used to study the specificity of the SPR biosensor, and the resultsshowed that the SPR biosensor had high combination with on the target bacteria, and very low non-target adsorption.
引文
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[7]March S B, Ratnam S. Sorbitol-Mac Conkey medium for detection of Escherichia coli 0157:H7 associated with hemorrhagic colitis. Clin. Microbiol.,1986,23: 869-872.
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[10]路昌峰.肠出血性大肠杆菌O157：H7的流行病学和实验诊断.中华综合医学,2001,2(11)：1042
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