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基于DNA传感器对硫酸盐还原菌检测的研究
zeng yan
学位类型博士
导师张盾
2016-05
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋腐蚀与防护
关键词硫酸盐还原菌 16s Rdna片段 Dna生物传感器 信号放大
摘要本论文以硫酸盐还原菌(SRB)为研究对象,探索了以特异性16S rDNA片段为目标检测物的DNA生物传感器的构建、优化及应用,分别构建了基于核酸酶循环放大技术的DNA生物传感器、基于溶菌酶信号探针的DNA生物传感器及基于DNA纳米生物条码-荧光系统的DNA生物传感器,实现了对SRB不同属种的高灵敏度检测,丰富了SRB的检测方法。主要的研究结果如下:
(1) 构建了基于核酸酶循环放大技术的DNA生物传感器。利用核苷酸分子的碱基互配原则,设计与目标基因片段互补的识别DNA探针和DNA信号探针,结合溶菌酶催化作用及核酸外切酶Ⅲ循环放大技术实现对目标物的检测。基于微生物特征遗传片段的检测技术具有极高的选择性,且对检测环境要求较低,能在复杂的环境体系下进行检测。
(2) 构建了基于溶菌酶信号探针的DNA生物传感器。以特异性16S rDNA片段为间接目标检测物,磁性微球为固相载体,结合单链DNA链作为捕获探针,溶菌酶DNA链作为信号探针,实现对微生物的定性及定量检测。该DNA传感器灵敏度高,荧光检测最低检测浓度达50 cfu mL-1, 同时在细菌的选择性检测及复杂溶液中具有显示了良好的应用性。
(3) 构建了基于DNA纳米生物条码-荧光系统的DNA生物传感器。以特异性16S rDNA片段为间接目标检测物,磁性微球为固相载体,结合单链DNA链作为捕获探针,DNA纳米生物条码作为信号探针,实现对微生物的定量检测,最低检测浓度达500 cfu mL-1。相比于之前类似方案,本检测系统具有很好的选择性及灵敏性,应用性广,进一步丰富了腐蚀微生物的检测方法。
其他摘要With Sulfate-reducing bacteria (SRB) as target, this thesis focus on the study for fabrication, optimization and application of DNA sensor. Detection methods including DNA sensor based on nuclease cycling amplification, bio-enzyme signal probe and DNA nanobarcode-fluorescence system were developed. All these methods are sensitive and selective, which has great scientific significance and potential practical application. The main results are show as follows:
1, By employing specific region amplicons of bacterial genome as target, genetic fragment detecting system is to design capture probe and signal probe complemented with specific target DNA. This study proposed a novel detecting method for bacterial DNA detection based on nuclease aided cycling amplification. This system has high selectivity, specificity and stability, and can be potentially applied in bacterial detection and cell analysis.
2, We described a simple and novel system for bacteria detection by using lysozyme as sensitive reporter. Coupled with target bacterial DNA binding with capture probe immobilized on MBs and lysozyme acted as signal reporter, this method is highly sensitive, with a minimum response concentration of 50 cfu mL-1 bacteria. In addition, the method exhibited excellent selectivity and applicability, being able to differentiate different bacteria target and used in complex buffer like human serum.
3, We designed a method of bacteria detection based on DNA nanobarcodes-based fluorescence system by flow cytometry. Couple with capture probe immobilized on MBs, lysozyme acted as signal reporter, fluorescencently-labeled DNA nanobarcode as novel signal labels, this method is both specific for bacteria and highly sensitive (down to 500 cfu mL-1 bacteria). Compared with traditional bacteria detecting methods mostly relied on the use of either small molecule affinity ligands or antibodies with some drawbacks like low sensitivity and limited bacteria application. This proposed nucleic acid-based approach allow specific quantifying of various bacteria species with 16S rRNA sequence information, emerging as a preferred technique for microbial identification over traditional biochemical assays
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/112534
专题海洋腐蚀与防护研究发展中心
作者单位中国科学院海洋研究所
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zeng yan. 基于DNA传感器对硫酸盐还原菌检测的研究[D]. 北京. 中国科学院大学,2016.
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