大肠杆菌O157:H7荧光微球免疫层析试纸条的研制

doi:10.7506/spkx1002-6630-201316072

食品科学

大肠杆菌O157:H7荧光微球免疫层析试纸条的研制

解泉源，赖卫华，刘春梅，孙吉昌，邓省亮，刘成伟，魏华，游兴勇，熊勇华

1.南昌大学食品科学与技术国家重点实验室，江西南昌 330047；2.无锡中德伯尔生物技术有限公司，江苏无锡 214000；
3.江西省疾病预防控制中心，江西南昌 330029；4.江西省科学院微生物研究所，江西南昌 330029

出版日期:2013-08-25 发布日期:2013-09-03

Development of Fluorescent Microsphere Immunochromatographic Strip for Detection of Escherichia coli O157:H7

XIE Quan-yuan，LAI Wei-hua，LIU Chun-mei，SUN Ji-chang，DENG Sheng-liang，LIU Cheng-wei，WEI Hua，YOU Xing-yong，XIONG Yong-hua

1. State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China；
2. Wuxi Zodolabs Biological Technology Co. Ltd., Wuxi 214000, China；
3. Jiangxi Province Center for Disease Control and Prevention, Nanchang 330029, China；
4. Institute of Microbiology, Jiangxi Academy of Sciences, Nanchang 330029, China

Online:2013-08-25 Published:2013-09-03

摘要/Abstract

摘要：

目的：建立快速、灵敏检测大肠杆菌O157:H7荧光微球免疫层析试纸条法。方法：以羧基化荧光微球作为标记物，共价偶联抗大肠杆菌O157:H7鼠源单克隆抗体，将大肠杆菌O157:H7兔多抗和驴抗鼠抗体喷涂于硝酸纤维素膜分别作为检测线和质控线，以双抗体夹心反应模式制备大肠杆菌O157:H7荧光微球免疫层析试纸条。结果：通过优化实验确定偶联缓冲液为0.02mol/L pH5.0磷酸盐缓冲液，抗体偶联微球量为100μg/mg，试纸条可在加样5min后判读结果。制备的荧光微球试纸条对纯培养大肠杆菌O157:H7进行检测，肉眼观察检测限在1.2×104CFU/mL，借助荧光读取仪检测限能提高到6.1×103CFU/mL。试纸条除与金黄色葡萄球菌有微弱交叉反应外，与实验室保存的30株细菌无交叉反应。结论：大肠杆菌O157:H7荧光微球试纸条具有操作简便、快速灵敏、易于量化的特点，为该菌的检测提供了一种新型的手段。

关键词: 大肠杆菌O157:H7, 荧光微球, 免疫层析法, 试纸条

Abstract:

Objectives: To develop a fluorescent microsphere immunochromatographic strip test for rapid and sensitive
detection of Escherichia coli O157:H7. Methods: Carboxyl-modified fluorescent microspheres were covalently coupled
with murine anti-E coli O157:H7 monoclonal antibody, and rabbit anti-E.coli O157:H7 polyclonal antibody and donkey
anti-mouse IgG were sprayed onto the nitrocellulose membrane as the test line and control line, respectively. The
fluorescent microsphere immunochromatographic strip for the detection of E.coli O157:H7 based on double antibody
sandwich ELISA (DAS-ELISA) was developed. Results: The coupling buffer used was 0.02 mol/L PB (pH 5.0). The
monoclonal antibody/microsphere ratio was 100 μg/mg. The procedure required only 5 min for detecting samples with the
test strip. The limit of naked eye detection was as low as 1.2 × 104 CFU/mL and could be increased to 6.1 × 103 CFU/mL
by the use of a fluorescence reader. No cross-reactivity with 30 laboratory strains was found despite marginal cross-reactivity
with Staphylococcus aureus. Conclusions: For E.coli O157:H7 detection, the fluorescent microsphere strip, which provided a
novel method, has the characteristics of user-friendly operation, rapidity, high sensitivity and ease of quantification.

Key words: Escherichia coli O157:H7, fluorescent microsphere, immunochromatography, strip

解泉源，赖卫华，刘春梅，孙吉昌，邓省亮，刘成伟，魏华，游兴勇，熊勇华. 大肠杆菌O157:H7荧光微球免疫层析试纸条的研制[J]. 食品科学, doi: 10.7506/spkx1002-6630-201316072.

XIE Quan-yuan，LAI Wei-hua，LIU Chun-mei，SUN Ji-chang，DENG Sheng-liang，LIU Cheng-wei，WEI Hua，YOU Xing-yong，XIONG Yong-hua. Development of Fluorescent Microsphere Immunochromatographic Strip for Detection of Escherichia coli O157:H7[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201316072.

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大肠杆菌O157:H7荧光微球免疫层析试纸条的研制

Development of Fluorescent Microsphere Immunochromatographic Strip for Detection of Escherichia coli O157:H7

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