以PCR为基础的BCG菌株基因分型及BCG DNA中CpG含量的测定

发布时间：2018-02-14 13:52

  本文关键词： 卡介苗 分型 聚合酶链反应 胞嘧啶-鸟嘌呤　出处：《华中科技大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:对目前国内卡介苗(BCG)生产用株进行基因分型,检测BCG DNA中胞嘧啶-鸟嘌呤(CpG)的含量。 方法:收集菌体分别经机械破碎或溶菌处理,用溴化十六烷基三甲基铵(CTAB)进行沉淀,再经酚:氯仿:异戊醇抽提,对所得DNA进行紫外扫描、凝胶电泳检测。主要采用PCR技术对11种BCG菌株及对比株提取的DNA进行体外扩增,引物针对BCG菌株某些特异性缺失及扩增染色体片段而设计,通过琼脂糖凝胶电泳检测扩增情况,并对所得产物进行测序鉴定。以CpG特异的甲基化酶(CpG Methylase)M.SssI对BCG DNA进行修饰,将CpG中的胞嘧啶(C)转化为5-甲基胞嘧啶(MC),利用限制性内切酶HpaⅡ进行消化以检测甲基化程度,甲基化完全的DNA经相关酶水解后应用反相高效液相色谱(RP-HPLC)方法检测样品中CpG的质量百分含量。 结果:机械破碎抽提BCG DNA产率高、纯度高,溶菌处理抽提DNA完整性好。在BCG相关片段的PCR扩增中,国内BCG生产用株均显示相同PCR结果,SenX3-regX3扩增片段为353 bp,RD Denmark/Glaxo扩增产物分别为281 bp(AL/AR)、1637 bp(BL/BR),nRD18扩增产物分别为655 bp(AL/AR)、406 bp(L/R/wtR),IS6110 copyⅡ扩增产物417 bp,RD2及nRD18(AL/CR)扩增无产物。BCG上海新菌种DNA样品的CpG质量百分含量为23.61%,巴斯德株抽提DNA样品的CpG质量百分含量为23.53%。 结论:CTAB沉淀是一种比较简便易行的BCG DNA提取方法。国内BCG生产用株均显示相同基因型,特异性缺失RD2,nRD18和RD Denmark/Glaxo均存在,IS6110为单拷贝,senX3-regX3基因间区域的77-bp分枝杆菌散在分布重复单位(MIRUs)为三重拷贝。在目前具有代表性的国际公认菌株中未能找到与国内株基因型匹配者。采用甲基化特异酶修饰、酶水解、RP-HPLC的方法,能检测出DNA样品中CpG的质量百分含量,而且这种方法的准确性得到了较好的验证。
[Abstract]:Aim: to detect the content of cytosine guanine guanine (CpG) in BCG DNA by genotyping of domestic BCG production strains. Methods: the bacteria were collected by mechanical crushing or bacteriolysis, precipitated by cetyltrimethylammonium bromide (CTAB), then extracted by phenol: chloroform: isoamyl alcohol. The DNA was scanned by UV. PCR technique was used to amplify DNA extracted from 11 BCG strains and control strains in vitro. Primers were designed for the specific deletion and amplification of chromosomal fragments of BCG strains. The amplification was detected by agarose gel electrophoresis. The BCG DNA was modified with CpG specific methylase CpG Methylase)M.SssI, and the cytosine cytosine in CpG was transformed into 5-methylcytosine, which was digested by restriction endonuclease Hpa 鈪，

本文编号：1510854