萊姆病螺旋體脈沖場凝膠電泳標準化技術建立及初步應用研究
發(fā)布時間：2018-07-21 19:07
【摘要】：研究背景： 脈沖場凝膠電泳PFGE是二十世紀八十年代由Schwartz和Canter發(fā)明的，后經(jīng)一系列發(fā)展，最終成為現(xiàn)代分子生物學中一項標準、規(guī)范的關鍵技術。 PFGE近年來被公認為細菌分型及追蹤傳染源最有效的方法之一，它能夠揭示完整的基因組遺傳特性。PFGE在細菌的研究中已普遍應用，特別是在疾病的暴發(fā)流行中，已經(jīng)顯示出了較強的應用價值。它不僅可單獨用于傳染病的追蹤溯源，而且也可輔助現(xiàn)場流行病調查，用以提供不同菌群間相互關聯(lián)的證據(jù)，現(xiàn)在已經(jīng)作為傳染病暴發(fā)中追蹤溯源不可缺少的有力工具。此外它還用于傳染病動態(tài)檢測，為預警提供重要參考信息。目前發(fā)達國家已建立起國際化病原菌分子分型實驗室監(jiān)測網(wǎng)絡（PulseNet），它是以嚴格的標準化的PFGE操作為基礎，通過網(wǎng)絡實驗室的檢測分析和相互間帶型的分析比較，建立數(shù)據(jù)交流的一個網(wǎng)絡平臺。 萊姆病在全球具有廣泛的分布，世界五大洲的七十多個國家已經(jīng)有萊姆病病例報告。我國是受萊姆病危害十分嚴重的國家，但目前尚未被列入法定報告?zhèn)魅静�。萊姆病的自然疫源地幾乎遍布我國所有山林地區(qū)，人群中有典型的萊姆病病例存在，我國受萊姆病威脅的人群接近1億人，估計我國萊姆病的每年新發(fā)病例不會少于2萬例。目前，絕大部分萊姆病患者得不到明確的診斷和有效的治療，40%～50%轉為中、晚期，長期反復發(fā)作，后果嚴重。 最近，研究發(fā)現(xiàn)萊姆病螺旋體的各個基因型與所導致疾病有很強相關性，即不同的基因型可以導致不同的疾病。在這種情況下，需要一種分型能力強、簡便、可靠、可以分辨不同萊姆病螺旋體基因型間與型內的分子生物學技術，用于萊姆病螺旋體的預防和控制。 本研究旨在將PFGE技術分型技術引入萊姆病螺旋體，建立萊姆病螺旋體PFGE的標準化操作流程SOP，進一步從染色體DNA角度分析萊姆病螺旋體分類特征，為遺傳學分類奠定基礎。 目前美國疾病預防控制中心（Centers for Disease Control and Prevention，CDC）及亞太地區(qū)PulseNet提供的其他病原體PFGE標準化操作程序，結合萊姆病螺旋體菌株的特性，對菌體基因組染色體DNA純化技術、限制性內切酶消解方案及脈沖場電泳參數(shù)進行優(yōu)化，初步建立萊姆病螺旋體脈沖場凝膠電泳技術，并對國際標準株和我國新疆、內蒙古、黑龍江、吉林、遼寧、河北、北京、四川、貴州等地菌株共56株進行聚類分析，獲得了高清晰度酶切圖譜，各株菌都有獨特的帶型分布，國內菌株與其標準菌株帶型相似，都具有優(yōu)勢基因型。不同基因型之間帶型完全不同。發(fā)現(xiàn)PFGE分型結果與RFLP和MLSA具有高度一致性。 目的： 本研究旨在建立并優(yōu)化萊姆病螺旋體的脈沖場電泳方法，進一步豐富和完善了萊姆病螺旋體的基因分型研究，為下一步開展萊姆病的預防控制及流行病學調查打下基礎。 方法： 利用目前美國CDC及亞太地區(qū)PulseNet提供的其他病原體PFGE標準化操作程序，結合萊姆病螺旋體菌株的特性，通過對萊姆病螺旋體基因組染色體DNA純化，限制性內切酶消解方案及脈沖場電泳參數(shù)進行優(yōu)化，初步建立萊姆病螺旋體脈沖場凝膠電泳技術，并對國際標準株和我國菌株進行聚類分析。 1、BSKII培養(yǎng)基的制備，稱量新蛋白胨、HEPES、酵母粉、丙酮酸鈉和1066等藥品，溶解后調節(jié)PH值，加入明膠、牛血清白蛋白Ⅴ和滅活的兔血清，采用硝酸纖維素膜過濾除菌，分裝備用； 2、采用BSKII培養(yǎng)基傳代培養(yǎng)購買自ATCC的國際標準菌株，并且從國內西北、東北等地區(qū)的人血標本、傳播媒介蜱和宿主動物鼠的標本中分離培養(yǎng)萊姆病螺旋體活菌，置于33℃恒溫箱。置菌夜于暗室野顯微鏡下觀察，待活菌生長繁殖到濃度+++至++++，才能制備膠塊，提取染色體DNA； 3、采用膠塊包埋法，將菌體包埋于膠塊中，裂解細菌，洗膠塊，膠塊內DNA的酶切，加樣，電泳，染色，獲取圖像，數(shù)據(jù)分析。選取34株萊姆病螺旋體，選擇不同的菌株濃度，不同的內切酶，不同的參數(shù)，重復上述實驗； 4、對得到的實驗圖片和數(shù)據(jù)進行分析，得到最佳的操作方案，最佳的菌株濃度，，最佳的內切酶和電泳參數(shù)； 5、再選取22株萊姆病螺旋體菌株，采用以上的出的最佳方案，得到聚類圖。分析聚類結果，對此實驗方法進行評價。 結果： 確立了萊姆病螺旋體PFGE分型方法的標準操作。 結論： 1、PFGE分型方法能較好的反映萊姆病菌株的分子遺傳學特征； 2、PFGE、RFLP和MLSA三種分型方法有較好的相關性； 3、PFGE分型方法受外界因素影響較小，可用于日常監(jiān)測菌株的鑒定分型。 創(chuàng)新點： 將PFGE技術分型技術引入萊姆病螺旋體，建立萊姆病螺旋體PFGE的標準化操作程序，確定萊姆病螺旋體PFGE分型方法的標準操作，進一步從染色體DNA角度分析萊姆病螺旋體分類特征，為遺傳學分類奠定基礎。在萊姆病的預防和控制工作中，提供標準化數(shù)據(jù)庫。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R514
本文編號：2136544
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R514
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