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为寻找食源性致病菌的快速定量检测方法,将阻抗技术和免疫层析技术相结合,研制一种双抗夹心免疫层析试纸条,探讨该试条电阻抗法定量检测大肠杆菌O157∶H7的可行性。采用具有良好导电性的聚苯胺标记大肠杆菌O157∶H7多克隆抗体,硝酸纤维素膜(NC膜)作为固相支撑物。通过阻抗-时间法表征层析过程中试条阻抗的变化,确定免疫反应稳定时间。通过交流阻抗法研究样品的多频响应,确定最佳测试频率。根据免疫反应稳定后不同浓度大肠杆菌O157∶H7样品相对空白样品的阻抗改变率,来实现对大肠杆菌O157∶H7的定量检测。试验结果表明,滴加样品180 s后免疫反应达到平衡。当测试频率为1Hz时,试纸条的阻抗变化率与菌液浓度的对数值呈线性相关。线性检测范围是1.15×103~1.15×106CFU/mL,决定系数R2为0.9616,最低检测限为7.08×102CFU/mL(S/N=3),检测时间低于6 min。该方法灵敏度高,检测时间短,操作简单,可应用于大肠杆菌O157∶H7的快速定量检测,同时为其它食源性致病菌的定量检测提供参考。
In order to find a fast and quantitative detection method of food-borne pathogens, the impedance technique and the immunochromatography technique were combined to develop a double-antibody sandwich immunoassay strip for the quantitative detection of E. coli O157 : H7’s feasibility. A polyaniline labeled E. coli O157: H7 polyclonal antibody with good conductivity and a nitrocellulose membrane (NC membrane) were used as the solid phase support. Impedance-time method was used to characterize the changes of the impedance of the strips during the chromatography to determine the stabilization time of the immunological reaction. The multi-frequency response of the sample was studied by AC impedance method to determine the best test frequency. According to the rate of change of the impedance of E. coli O157: H7 samples relative to blank samples after the immune response was stabilized, the quantitative detection of E. coli O157: H7 was achieved. The experimental results show that the immune response is balanced after 180 s of dropping. When the test frequency is 1Hz, the impedance change rate of the strip is linearly related to the logarithm of the concentration of the bacterial solution. The linear detection range was 1.15 × 103 ~ 1.15 × 106CFU / mL. The determination coefficient R2 was 0.9616. The detection limit was 7.08 × 102CFU / mL (S / N = 3). The detection time was less than 6 min. The method has high sensitivity, short detection time and simple operation, and can be applied to the rapid and quantitative detection of Escherichia coli O157: H7 and provides a reference for the quantitative detection of other foodborne pathogens.