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 陶晴 卞晓军 陈谦 颜娟

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

    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。

English


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  • 收稿日期:  2020-12-08
  • 修回日期:  2021-01-13
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