基于滚环扩增技术的DNA水凝胶用于大肠杆菌O157:H7的可视化快速检测

引用本文: 张彤, 陶晴, 卞晓军, 陈谦, 颜娟. 基于滚环扩增技术的DNA水凝胶用于大肠杆菌O157:H7的可视化快速检测[J]. 分析化学, 2021, 49(3): 377-386. doi: 10.19756/j.issn.0253-3820.201744 shu

Citation: ZHANG Tong, TAO Qing, BIAN Xiao-Jun, CHEN Qian, YAN Juan. Rapid Visualized Detection of Escherichia Coli O157: H7 by DNA Hydrogel Based on Rolling Circle Amplification[J]. Chinese Journal of Analytical Chemistry, 2021, 49(3): 377-386. doi: 10.19756/j.issn.0253-3820.201744 shu

基于滚环扩增技术的DNA水凝胶用于大肠杆菌O157:H7的可视化快速检测

张彤 ^1, ,
陶晴 ^1, ,
卞晓军 ^1,2,3, ,
陈谦 ^1, ,
颜娟 ^{1,2,3,
,}

1.
上海海洋大学食品学院, 上海 201306;
2.
上海水产品加工及贮藏工程技术研究中心, 上海 201306;
3.
农业部水产品贮藏保鲜质量安全风险评估实验室(上海), 上海 201306

通讯作者: 颜娟,E-mail:j-yan@shou.edu.cn

基金项目:
国家自然科学基金项目（No.21775102）和上海市自然科学基金项目（No.20ZR1424100）资助。

摘要: 以大肠杆菌O157：H7（Escherichia coli O157：H7，E.coli O157：H7）为研究模式菌，构建了基于醛基化磁珠（Aldehyde magnetic beads，AMBs）、滚环扩增技术（Rolling circle amplification，RCA）和DNA水凝胶可视化快速检测的适配体生物传感器（Aptasensor）用于食品安全检测。制备了Mbs@dsDNA （Double-stranded DNA，dsDNA）杂交复合物，当待测物中含有E.coli O157：H7时，其与磁珠表面的适配体序列发生特异性识别，将含有引发序列的适配体链释放至上清液中。通过磁响应分离收集上清液，上清液中的适配体引发探针与成环序列杂交形成挂锁探针，添加T4 DNA连接酶、phi 29 DNA聚合酶和dNTPs等引发RCA；当待测物中不含E.coli O157：H7时，无法引发RCA。本研究使用两条成环序列，其部分碱基互补。这两条成环序列的挂锁探针经过RCA后生成两条可部分互补杂交的长单链DNA （Single-stranded DNA，ssDNA）产物，两管ssDNA产物振荡孵育，可形成裸眼可见的DNA水凝胶，从而实现E.coli O157：H7的可视化检测。本方法可在1 h内实现最低浓度为4×10³ CFU/mL的E.coli O157：H7的可视化检测，检测时间低至30 min，具有较高的灵敏度和特异性，且操作简便、快速，不需要大型仪器，在食品安全检测方面具有潜在的应用前景。

关键词:

English

Rapid Visualized Detection of Escherichia Coli O157: H7 by DNA Hydrogel Based on Rolling Circle Amplification

1.
College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China;
2.
Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai 201306, China;
3.
Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai 201306, China

Corresponding author: YAN Juan, j-yan@shou.edu.cn

Received Date: 08 December 2020
Revised Date: 13 January 2021
Fund Project:
National Natural Science Foundation of China (No. 21775102) and the Natural Science Foundation of Shanghai Municipal, China (No. 20ZR1424100)

Abstract: Foodborne illnesses caused by pathogenic bacteria are prominent issues in food safety. Fast and sensitive detection of pathogenic bacteria is significantly essential for food safety. In this work, an aptasensor based on aldehyde magnetic beads, rolling circle amplification (RCA) and DNA hydrogel was developed for visualized, simple and rapid detection of Escherichia Coli O157:H7 (E.coli O157:H7). The Mbs@dsDNA complexes were first prepared. In the presence of target, the aptamer combined with E.coli O157:H7, which released E.coliaptamer-initiator. The supernatant collected by magnetic separation and the released primers were hybridized with circular sequence to form padlock probe. Then, the RCA reaction was initiated by adding T4 DNA ligase, phi29 DNA polymerase and dNTPs, while the RCA could not be triggered in the absence of target. Those two circular sequences were designed to contain partially complementary bases. Thus, when those two circular sequences generated two long single-stranded DNA (ssDNA) products respectively after RCA reaction, two ssDNA products hybridized with each other and formed naked-eye visible DNA hydrogel. The aptasensor was successfully used for visualized detection of E.coli O157:H7 with a detection limit of 4×10³ CFU/mL, and the detection could be finished within 30 min. The aptasensor had many advantages such as high specificity and specificity, easy operation, efficient amplification, and rapid and visualized readout, showing potential in applications of food safety detection.

Key words:

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沈阳化工大学材料科学与工程学院沈阳 110142

基于滚环扩增技术的DNA水凝胶用于大肠杆菌O157:H7的可视化快速检测

通讯作者: 颜娟,E-mail:j-yan@shou.edu.cn

English

Rapid Visualized Detection of Escherichia Coli O157: H7 by DNA Hydrogel Based on Rolling Circle Amplification

Corresponding author: YAN Juan, j-yan@shou.edu.cn

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

基于滚环扩增技术的DNA水凝胶用于大肠杆菌O157:H7的可视化快速检测

通讯作者: 颜娟,E-mail:j-yan@shou.edu.cn

English

Rapid Visualized Detection of Escherichia Coli O157: H7 by DNA Hydrogel Based on Rolling Circle Amplification

Corresponding author: YAN Juan, j-yan@shou.edu.cn

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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