Rapid, species-specific detection of uropathogen 16S rDNA and rRNA at ambient temperature by dot-blot hybridization and an electrochemical sensor array
Rapid, species-specific detection of uropathogen 16S rDNA and rRNA at ambient temperature by dot-blot hybridization and an electrochemical sensor array
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DOI：
10.1016/j.ymgme.2004.11.006
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发表时间：
2005-01-01
影响因子：
3.8
通讯作者：
Haake, DA
中科院分区：
文献类型：
--
作者：
Sun, CP;Liao, JC;Haake, DA
发展用于病原体检测的快速分子方法是改进传染病治疗的关键。在这项研究中，对尿路病原体物种特异性序列的DNA探针杂交的动力学和温度依赖性进行了研究。根据大肠埃希菌、奇异变形杆菌、催产克雷伯氏菌和铜绿假单胞菌的16S基因可变区，设计了一套寡核苷酸探针。还包括一种通用的细菌探针和专用于革兰氏阳性和革兰氏阴性生物的探针。该寡核苷酸探针以斑点印迹的形式区分了来自不同种类的泌尿系病原菌的16S基因。寡核苷酸探针与靶DNA的显著结合和膜清洗去除非特异性结合均可快速实现，仅需10分钟。在基于微制造技术的新型电化学16传感器阵列中，使用来自E.Coli16S基因同一物种特异区的寡核苷酸探针作为捕获探针。用辣根过氧化物酶作为电化学传感器，通过第二个探测器探针，检测目标尿路病原菌16S rDNA的序列特异性杂交。该传感器阵列在与斑点杂交研究的快速动力学一致的时间过程中显示出快速的、物种特异性的杂交。正如在斑点杂交研究中一样，使用传感器阵列方法在65摄氏度和室温下都证明了细菌核酸的物种特异性检测。这些结果表明，分子杂交方法可以适应快速的室温条件，是电化学传感器阵列平台的理想选择。由爱思唯尔公司出版。
Development of rapid molecular approaches for pathogen detection is key to improving treatment of infectious diseases. For this study, the kinetics and temperature-dependence of DNA probe hybridization to uropathogen species-specific sequences were examined. A set of oligonucleotide probes were designed based on variable regions of the 16S gene of the Escherichia coli, Proteus mirabilis, Klebsiella oxytoca, and Pseudomonas aeruginosa. A universal bacterial probe and probes-specific for gram-positive and gram-negative organisms were also included. The oligonucleotide probes discriminated among 16S genes derived from I I different species of uropathogenic bacteria applied to nylon membranes in a dot-blot format. Significant binding of oligonucleotide probes to target DNA and removal of nonspecific binding by membrane washing could both be achieved rapidly, requiring as little as 10 min. An oligonucleotide probe from the same species-specific region of the E coli 16S gene was used as a capture probe in a novel electro-chemical 16-sensor array based on micro fabrication technology. Sequence-specific hybridization of target uropathogen 16S rDNA was detected through horseradish peroxidase acting as an electrochemical transducer via a second, detector probe. The sensor array demonstrated rapid, species-specific hybridization in a time course consistent with the rapid kinetics of the dot-blot hybridization studies. As in the dot-blot hybridization studies, species-specific detection of bacterial nucleic acids using the sensor array approach was demonstrated both at 65 degreesC and at room temperature. These results demonstrate that molecular hybridization approaches can be adapted to rapid, room temperature conditions ideal for an electrochemical sensor array platform. Published by Elsevier Inc.