基于16SrDNA多态性的细菌鉴定寡核苷酸芯片的初步研究

发布时间：2018-03-23 21:55

本文选题：16SrDNA　切入点：病原性细菌　出处：《第三军医大学》2005年硕士论文

【摘要】：背景致病菌的快速检测是临床微生物学诊断迫切需要解决的一个难题;目前,临床细菌学检测仍主要依赖基于观察细菌形态的检查、基于分析细菌代谢特性的生化试验和基于检测特定抗原或抗体的免疫血清学方法。生化鉴定需要对细菌进行分离培养,耗时长(3～7d);血清学试验在感染初期往往是阴性;16SrDNA 序列的变异能够反映细菌的种属关系,已经用于细菌的分类研究中;基于PCR 的基因诊断技术已广泛应用于临床病原体的诊断,单纯的PCR 诊断特异性差,假阳性多见,核酸杂交检测能较好地克服这一问题;基因芯片是一种高通量、特异、快速的核酸杂交检测技术。将PCR 和基因芯片两种技术结合在一起,则可能实现对致病菌的快速检测。本研究的目的是探讨应用16SrDNA 检测临床常见感染性细菌的可行性。目的建立灵敏、特异的检测16SrDNA 多态性的寡核苷酸芯片的技术路线。方法 1、利用通用引物扩增部分常见病原性细菌的16SrDNA,并克隆、测序,以用做标准阳性对照。 2、收集RDP-II 数据库中的临床常见细菌的16SrDNA 的序列信息,结合我们克隆的标准对照的测序结果设计相应的探针。 3、片段化杂交用的PCR 扩增产物。 4、利用PCR 方法标记待检测的靶序列,与玻片表面的探针进行固相杂交。结果 1、利用筛选的通用引物,成功克隆了22株常见细菌的近全长(约1.5kb)的16SrDNA基因。 2、找到了一种在U(T)碱基处特异性片段化PCR 产物的方法。 3、通过优化探针设计、杂交条件、产物高效标记与片段化等方法,初步制备出可以检测细菌16SrDNA 寡核苷酸芯片并建立了相应的检测方法。
[Abstract]:Background the rapid detection of pathogenic bacteria is an urgent problem to be solved in clinical microbiological diagnosis. At present, clinical bacteriological detection is still mainly based on the observation of bacterial morphology. Biochemical tests based on the analysis of metabolic characteristics of bacteria and immune serological methods based on the detection of specific antigens or antibodies. Biochemical identification requires isolation and culture of bacteria, The variation of 16s rDNA sequence in serological tests is often negative in the early stage of infection, which can reflect the species relationship of bacteria and has been used in the taxonomy of bacteria. The gene diagnosis technology based on PCR has been widely used in the diagnosis of clinical pathogens. Simple PCR has poor specificity and false positivity, which can be overcome by nucleic acid hybridization. Gene chip is a high throughput, specific and fast nucleic acid hybridization technique. PCR and gene chip are combined together. The aim of this study was to explore the feasibility of 16SrDNA for the detection of common clinical infectious bacteria. Purpose. To establish a sensitive and specific oligonucleotide chip for the detection of 16SrDNA polymorphism. Method. 1. The 16s rDNA of some common pathogenic bacteria was amplified by universal primers, cloned and sequenced to be used as standard positive control. 2. The 16SrDNA sequences of common clinical bacteria in RDP-II database were collected, and the corresponding probes were designed based on the results of our cloned standard control sequencing. 3, PCR amplification products for fragment hybridization. (4) PCR method was used to label the target sequence to be detected, and a solid phase hybridization was carried out with the probe on the glass surface. Results. 1. The nearly full-length (1.5 kb) 16SrDNA gene of 22 strains of common bacteria was successfully cloned by using universal primers. 2. A method for specific fragmentation of PCR products at UT base was found. 3. By optimizing the probe design, hybridization conditions, high efficiency labeling and fragmentation of the product, the microarray of bacterial 16SrDNA oligonucleotide was prepared and the corresponding detection method was established.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R378

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