化脓性脑膜炎脑脊液致病菌高通量测序及物种分析

发布时间：2018-05-13 09:12

本文选题：化脓性脑膜炎 + 脑脊液　；参考：《宁夏医科大学》2013年硕士论文

【摘要】：目的研究化脓性脑膜炎患者脑脊液中致病菌的常见细菌种类、细菌丰度及菌群结构，探讨化脓性脑膜炎病原学诊断的新途径和方法。方法本研究选取临床诊断为化脓性脑膜炎患者脑脊液样本61例，提取化脓性脑膜炎脑脊液细菌基因组DNA、再进行PCR鉴定，同时对CSF进行细菌培养，计算两种方法的阳性率，将二者阳性率采用SPSS17.0统计软件进行卡方检验。利用16S rDNA宏基因组测序技术对PCR阳性的样本进行高通量测序，通过生物信息学分析的方法对测序结果进行整合、分析。结果对选取的61例化脓性脑膜炎脑脊液样本分别进行细菌培养和检验，有26例样本在550bp处扩增出特异的DNA产物，而细菌培养阳性的样本仅为10例，61例脑脊液样本中，26例16S rDNA PCR阳性，，阳性率为42.6%；61例脑脊液样本中细菌培养阳性为10例，阳性率为16.4%；通过16S rDNA宏基因组测序技术及生物信息学技术研究发现：通过观察每个样品所含有的OTU数量，与其他环境相比，脑脊液含有的物种种类相对偏少；PM脑脊液样本致病菌的种类大致可以分为以厚壁菌门和变形杆菌门为主，以属为分类标准，优势菌属主要为链球菌属，鲍曼不动杆菌，假单胞菌，奈瑟菌属等；通过beta多样性分析, PM脑脊液中细菌感染在属的水平上两两样本之间差异不大。结论通过对上述两种检测方法阳性率比较，PCR检查方法诊断化脓性脑膜炎明显优于分离培养法（P＜0.05）；本研究选取QIAGEN DNeasy Blood and Tissue Kit试剂盒DNA提取的方法，获得的基因组DNA，引物及PCR条件较好，能对化脓性脑膜炎脑脊液样本中16S rDNA进行良好的扩增，能适用于化脓性脑膜炎脑脊液宏基因组学的后续数据分析及研究。通过16S rDNA宏基因组测序技术对于化脓性脑膜炎脑脊液致病菌的种类，丰度及菌群结构的分析发现银川地区感染的优势菌群为：链球菌，鲍曼不动杆菌及假单胞菌，还有一些未知的细菌也是造成颅内感染的重要元凶。
[Abstract]:Objective to study the common bacteria species, bacterial abundance and bacterial community structure in cerebrospinal fluid (CSF) of patients with suppurative meningitis, and to explore a new approach and method for etiological diagnosis of suppurative meningitis. Methods in this study, 61 samples of cerebrospinal fluid (CSF) from patients with suppurative meningitis were selected to extract bacterial genomic DNA from cerebrospinal fluid (CSF) of suppurative meningitis, then PCR was used to identify CSF, and the positive rate of the two methods was calculated. The positive rates were tested by SPSS17.0 software. High-throughput sequencing of PCR positive samples was carried out by using 16s rDNA macro genome sequencing technique. The results were integrated and analyzed by bioinformatics analysis. Results bacterial culture and examination were carried out in 61 samples of cerebrospinal fluid from suppurative meningitis. Specific DNA products were amplified from 26 samples at 550bp. The positive rate of bacterial culture was 42.6%. The positive rate of bacterial culture was 42.6%, and the positive rate of bacterial culture was 10 cases in 61 cerebrospinal fluid samples. The positive rate was 16. 4%. By studying 16s rDNA macro genome sequencing and bioinformatics, we found that by observing the amount of OTU in each sample, compared with other environments, The species contained in cerebrospinal fluid (CSF) were relatively few. The species of pathogenic bacteria in CSF samples of PM could be roughly divided into two groups: phylum platyphylum and phylum Proteus. The dominant species were Streptococcus and Acinetobacter baumannii. Pseudomonas, Neisseria and so on. By beta diversity analysis, bacterial infection in PM cerebrospinal fluid had little difference between two samples at the generic level. Conclusion the positive rate of the above two methods is better than that of the culture method (P < 0.05) in the diagnosis of suppurative meningitis, and the method of DNA extraction with QIAGEN DNeasy Blood and Tissue Kit kit is selected in this study. The obtained genomic DNA, primers and PCR conditions were good for amplification of 16s rDNA in cerebrospinal fluid samples of suppurative meningitis, and suitable for subsequent data analysis and research on macrogenomics of cerebrospinal fluid in suppurative meningitis. By analyzing the species, abundance and microflora structure of cerebrospinal fluid pathogenic bacteria of suppurative meningitis by 16s rDNA macro genome sequencing, it was found that the dominant bacterial groups of infection in Yinchuan area were Streptococcus, Acinetobacter baumannii and Pseudomonas. Unknown bacteria are also responsible for intracranial infections.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R515.2

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