Electrochemical detection of PCR amplicons of Escherichia coli genome based on DNA nanostructural probes and polyHRP enzyme
Electrochemical detection of PCR amplicons of Escherichia coli genome based on DNA nanostructural probes and polyHRP enzyme
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基于DNA纳米结构探针和polyHRP酶的大肠杆菌基因组PCR扩增子的电化学检测
DOI：
10.1039/c6an01435f
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发表时间：
2016-01-01
期刊：
影响因子：
4.2
通讯作者：
Zuo, Xiaolei
中科院分区：
文献类型：
--
作者：
Wen, Yanli;Wang, LeLe;Zuo, Xiaolei
快速、便携、灵敏的E.大肠杆菌正在成为许多关键领域的重要挑战（例如，食品安全、环境监测和临床诊断）。因此，来自细菌基因组的PCR扩增子的电化学生物传感引起了合理的研究关注。本工作利用三维DNA四面体探针构建了一种“双螺旋型”电化学DNA生物传感器，用于检测大肠杆菌uidA基因的250 bp未纯化PCR扩增子。大肠杆菌基因组。不对称PCR用于产生单链PCR产物。采用链霉亲和素-聚HRP 80提高电化学检测的信号增益。优化了DNA传感的实验条件，包括链霉亲和素-聚HRP，信号探针和离子强度。最后，我们获得了显着的灵敏度为10 fM的合成DNA的目标，并成功地进行了PCR扩增子分析低至0.2 pg μ L-1的E。大肠杆菌基因组。与传统的基于单链DNA（ssDNA）探针的检测相比，本工作的灵敏度提高了3个数量级。此外，我们的电化学DNA生物传感器是4个数量级以上的灵敏度比正常的PCR产物的电泳分析。我们的工作使基于DNA纳米结构探针的生物传感器在走向真实的应用方面取得了重要进展。
Fast, portable and sensitive analysis of E. coli is becoming an important challenge in many critical fields (e.g., food safety, environmental monitoring and clinical diagnosis). Thus, electrochemical biosensing of PCR amplicons from the bacterial genome has attracted reasonable research attention. In this work, we utilized a 3D DNA tetrahedral probe to establish a "sandwich-type" electrochemical DNA biosensor for sensitive and specific analysis of a 250 bp unpurified PCR amplicon from the uidA gene of the E. coli genome. Asymmetric PCR was used to produce single-stranded PCR products. Streptavidin-polyHRP80 was employed to improve the signal gain during electrochemical detection. We optimized important experimental conditions for DNA sensing, including the streptavidin-polyHRP, the signal probe and the ion strength. Finally, we achieved a remarkable sensitivity of 10 fM synthetic DNA target, and successfully performed the analysis of PCR amplicons from as low as 0.2 pg mu L-1 of E. coli genome. Compared with traditional single stranded DNA (ssDNA) probe based detection, our present work demonstrated 3 orders of magnitude improvement in sensitivity. In addition, our electrochemical DNA biosensor was 4 orders of magnitude more sensitive than normal electrophoretic analysis of PCR products. Our work made important progress in DNA nanostructured probe-based biosensors toward application in real applications.