莱姆病螺旋体脉冲场凝胶电泳标准化技术建立及初步应用研究

发布时间：2018-07-21 19:07

【摘要】：研究背景：脉冲场凝胶电泳PFGE是二十世纪八十年代由Schwartz和Canter发明的，后经一系列发展，最终成为现代分子生物学中一项标准、规范的关键技术。 PFGE近年来被公认为细菌分型及追踪传染源最有效的方法之一，它能够揭示完整的基因组遗传特性。PFGE在细菌的研究中已普遍应用，特别是在疾病的暴发流行中，已经显示出了较强的应用价值。它不仅可单独用于传染病的追踪溯源，而且也可辅助现场流行病调查，用以提供不同菌群间相互关联的证据，现在已经作为传染病暴发中追踪溯源不可缺少的有力工具。此外它还用于传染病动态检测，为预警提供重要参考信息。目前发达国家已建立起国际化病原菌分子分型实验室监测网络（PulseNet），它是以严格的标准化的PFGE操作为基础，通过网络实验室的检测分析和相互间带型的分析比较，建立数据交流的一个网络平台。莱姆病在全球具有广泛的分布，世界五大洲的七十多个国家已经有莱姆病病例报告。我国是受莱姆病危害十分严重的国家，但目前尚未被列入法定报告传染病。莱姆病的自然疫源地几乎遍布我国所有山林地区，人群中有典型的莱姆病病例存在，我国受莱姆病威胁的人群接近1亿人，估计我国莱姆病的每年新发病例不会少于2万例。目前，绝大部分莱姆病患者得不到明确的诊断和有效的治疗，40%～50%转为中、晚期，长期反复发作，后果严重。最近，研究发现莱姆病螺旋体的各个基因型与所导致疾病有很强相关性，即不同的基因型可以导致不同的疾病。在这种情况下，需要一种分型能力强、简便、可靠、可以分辨不同莱姆病螺旋体基因型间与型内的分子生物学技术，用于莱姆病螺旋体的预防和控制。本研究旨在将PFGE技术分型技术引入莱姆病螺旋体，建立莱姆病螺旋体PFGE的标准化操作流程SOP，进一步从染色体DNA角度分析莱姆病螺旋体分类特征，为遗传学分类奠定基础。目前美国疾病预防控制中心（Centers for Disease Control and Prevention，CDC）及亚太地区PulseNet提供的其他病原体PFGE标准化操作程序，结合莱姆病螺旋体菌株的特性，对菌体基因组染色体DNA纯化技术、限制性内切酶消解方案及脉冲场电泳参数进行优化，初步建立莱姆病螺旋体脉冲场凝胶电泳技术，并对国际标准株和我国新疆、内蒙古、黑龙江、吉林、辽宁、河北、北京、四川、贵州等地菌株共56株进行聚类分析，获得了高清晰度酶切图谱，各株菌都有独特的带型分布，国内菌株与其标准菌株带型相似，都具有优势基因型。不同基因型之间带型完全不同。发现PFGE分型结果与RFLP和MLSA具有高度一致性。目的：本研究旨在建立并优化莱姆病螺旋体的脉冲场电泳方法，进一步丰富和完善了莱姆病螺旋体的基因分型研究，为下一步开展莱姆病的预防控制及流行病学调查打下基础。方法：利用目前美国CDC及亚太地区PulseNet提供的其他病原体PFGE标准化操作程序，结合莱姆病螺旋体菌株的特性，通过对莱姆病螺旋体基因组染色体DNA纯化，限制性内切酶消解方案及脉冲场电泳参数进行优化，初步建立莱姆病螺旋体脉冲场凝胶电泳技术，并对国际标准株和我国菌株进行聚类分析。 1、BSKII培养基的制备，称量新蛋白胨、HEPES、酵母粉、丙酮酸钠和1066等药品，溶解后调节PH值，加入明胶、牛血清白蛋白Ⅴ和灭活的兔血清，采用硝酸纤维素膜过滤除菌，分装备用； 2、采用BSKII培养基传代培养购买自ATCC的国际标准菌株，并且从国内西北、东北等地区的人血标本、传播媒介蜱和宿主动物鼠的标本中分离培养莱姆病螺旋体活菌，置于33℃恒温箱。置菌夜于暗室野显微镜下观察，待活菌生长繁殖到浓度+++至++++，才能制备胶块，提取染色体DNA； 3、采用胶块包埋法，将菌体包埋于胶块中，裂解细菌，洗胶块，胶块内DNA的酶切，加样，电泳，染色，获取图像，数据分析。选取34株莱姆病螺旋体，选择不同的菌株浓度，不同的内切酶，不同的参数，重复上述实验； 4、对得到的实验图片和数据进行分析，得到最佳的操作方案，最佳的菌株浓度，，最佳的内切酶和电泳参数； 5、再选取22株莱姆病螺旋体菌株，采用以上的出的最佳方案，得到聚类图。分析聚类结果，对此实验方法进行评价。结果：确立了莱姆病螺旋体PFGE分型方法的标准操作。结论： 1、PFGE分型方法能较好的反映莱姆病菌株的分子遗传学特征； 2、PFGE、RFLP和MLSA三种分型方法有较好的相关性； 3、PFGE分型方法受外界因素影响较小，可用于日常监测菌株的鉴定分型。创新点：将PFGE技术分型技术引入莱姆病螺旋体，建立莱姆病螺旋体PFGE的标准化操作程序，确定莱姆病螺旋体PFGE分型方法的标准操作，进一步从染色体DNA角度分析莱姆病螺旋体分类特征，为遗传学分类奠定基础。在莱姆病的预防和控制工作中，提供标准化数据库。
[Abstract]:Research background:
Pulsed field gel electrophoresis (PFGE) was invented by Schwartz and Canter in 1980s. After a series of developments, it became a standard and key technology in modern molecular biology.
PFGE has been recognized as one of the most effective methods for bacterial typing and tracing the source of infection in recent years. It can reveal that the complete genomic genetic characteristics,.PFGE, have been widely used in the study of bacteria, especially in the outbreak of disease, which has shown strong application value. It can be used not only to trace the traceability of infectious diseases, but also to be used alone. It can also assist the field epidemiological survey to provide evidence of interrelated strains between different bacteria groups. It has now been used as an indispensable tool for tracing the origin of infectious diseases. In addition, it is also used for dynamic detection of infectious diseases and provides important reference information for early warning. At present, the developed countries have established molecular typing experiments of international pathogens. Room monitoring network (PulseNet), which is based on strict standardized PFGE operations, establishes a network platform for data communication through the detection analysis of network laboratories and analysis and comparison with each other.
Lyme disease is widely distributed throughout the world. More than 70 countries in the five continents have reported cases of Lyme disease. China is a country affected by Lyme disease, but it has not been included in the legal report of infectious disease. The natural foci of Lyme disease are almost all over the mountain areas in our country, and there are typical Lyme disease among the population. There are 100 million people threatened by Lyme disease in our country. It is estimated that there will be no less than 20 thousand new cases of Lyme disease in China each year. At present, most Lyme disease patients have no definite diagnosis and effective treatment, 40% to 50% turn into middle, late, long-term recurrent attacks and serious consequences.
Recent studies have found a strong correlation between the various genotypes of Lyme disease spirals and the causes of disease, that is, different genotypes can lead to different diseases. In this case, a strong, simple and reliable type of typing is needed to distinguish the molecular biological techniques of different Lyme disease helix based and intra type, used for Lyme disease. Prophylaxis and control of spirals.
The purpose of this study is to introduce PFGE technology into Lyme disease spirals and establish the standardized operation process of Lyme disease spirals PFGE, SOP, to further analyze the classification characteristics of Lyme disease spirals from the angle of chromosome DNA, and lay the foundation for genetic classification.
At present, the Centers for Disease Control and Prevention, CDC, and other pathogen PFGE standardized operation procedures provided by PulseNet in the Asia Pacific region, combined with the characteristics of Lyme helix strain, the purification of the genomic chromosome DNA of the mycelium, the restriction endonuclease digestion scheme and the parameters of the pulse field electrophoresis. On the basis of optimization, a preliminary establishment of Lyme disease spiral pulse field gel electrophoresis (pdgge) was established, and 56 strains of strains in Xinjiang, Inner Mongolia, Heilongjiang, Jilin, Liaoning, Hebei, Beijing, Sichuan and Guizhou were clustered and analyzed. The high definition enzyme cut atlas was obtained. The quasi bacterial strains were similar in type. They all had the dominant genotype. The different genotype types were completely different. The results of PFGE typing were highly consistent with those of RFLP and MLSA.
Objective:
The purpose of this study is to establish and optimize the pulse field electrophoresis of Lyme disease spirals, to further enrich and improve the genotyping of Lyme disease spirals, and to lay a foundation for the next step in the prevention and control of Lyme disease and epidemiological investigation.
Method:
Using the standardized operating procedures of other pathogens PFGE provided by the American CDC and PulseNet in the Asia Pacific region, and combining the characteristics of Lyme disease spirulum, by purifying the genomic chromosome DNA of Lyme disease spirulum genome, optimizing the restriction endonuclease digestion scheme and pulse field electrophoresis parameters, the preliminary establishment of Lyme disease spiral pulse field coagulation is preliminarily established. Gel electrophoresis technology, and cluster analysis of international standard strains and Chinese strains.
1, the preparation of BSKII medium, weighing new peptone, HEPES, yeast powder, sodium pyruvate and 1066, and regulating pH value, adding gelatin, bovine serum albumin V and inactivated rabbit serum, using nitrocellulose membrane filtration to remove bacteria and equipment;
2, the BSKII culture medium was used to culture the international standard strains from ATCC, and from the human blood specimens of the northwest and northeastern regions, the strains of Lyme disease spirals were isolated and cultured from the specimens of the media ticks and the host animals. The bacteria were placed at the constant temperature box at 33 degrees C. The bacteria were observed under the dark room microscope, and the living bacteria were grown to the concentration + + +. In addition to + + +, glue block can be prepared and chromosome DNA is extracted.
3, using the glue mass embedding method, the bacteria were embedded in the glue block, lysis bacteria, gum washing block, and the enzyme of DNA in glue block, sample, electrophoresis, dyeing, obtain images, data analysis. Select 34 Lyme disease spirals, select different strains concentration, different endonuclease, different parameters, repeat the above experiments;
4, analyze the experimental pictures and data, get the best operation plan, the best strain concentration, the best endonuclease and electrophoresis parameters.
5, 22 strains of Borrelia burgdorferi were selected, and the best plan was selected to get the cluster map. The clustering results were analyzed and the experimental method was evaluated.
Result:
Standard operation of Lyme disease helicoid PFGE typing method was established.
Conclusion:
1, PFGE typing method can better reflect the molecular genetic characteristics of Lyme strains.
2, there is a good correlation between three typing methods of PFGE, RFLP and MLSA.
3, the PFGE typing method is less affected by external factors, and can be used for identification and typing of routine monitoring strains.
Innovation point:
The PFGE technology classification technique was introduced to Lyme disease spirals and the standardized operation procedure of Lyme disease spirals PFGE was established. The standard operation of Lyme disease spirals PFGE typing method was determined. The classification characteristics of Lyme disease spirals were further analyzed from the angle of chromosome DNA, and the basis for genetic classification was laid. Provide a standardized database.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R514

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