结核分枝杆菌环介导等温扩增实时荧光与反向斑点杂交检测方法的研究
发布时间：2018-07-22 15:23
【摘要】：结核病是一种严重威胁人类健康的传染病，是我国重点防控的疾病之一，也是全世界重点关注的公共卫生和社会问题。据世界卫生组织（WHO）统计，全球目前约有860万人罹患结核病，130万人死于结核病。结核病的发病率以平均每年增加0.4%的速度增长。因此，寻找一种快速准确的结核分枝杆菌检测方法已刻不容缓。近年来兴起的环介导等温扩增技术具有快速、敏感性高、特异性强等优点，而反向斑点杂交技术结合了基因扩增与分子杂交两项技术，可准确、直观的观察结果，操作简便。本研究建立了结核分枝杆菌环介导等温扩增技术和反向斑点杂交技术，可以满足现场快速检测的需求，为结核病的防控提供了技术支持。 本研究分为四部分： 第一部分：结核分枝杆菌插入序列IS6110基因的克隆与序列分析 选择结核分枝杆菌特异性较好的插入序列IS6110片段作为靶序列，根据GenBank上公布的的结核分枝杆菌IS6110序列(GenBank登录号为:X17348.1)设计一对特异性引物。将PCR扩增后的目的片段与pGEM-T载体相连接，连接产物转化至大肠杆菌感受态细胞，通过蓝白斑筛选得到克隆质粒pGEM-T-IS6110。克隆质粒经PCR及测序鉴定，结果证实获得了结核分枝杆菌IS6110基因。 第二部分：结核分枝杆菌环介导等温扩增实时荧光检测方法的建立 针对结核分枝杆菌特有的插入序列IS6110，自行设计并合成结核分枝杆菌的环介导等温扩增引物组(包括一对外引物以及一对内引物)，，将引物组中的各引物按照一定的摩尔浓度比例配制成引物组溶液，并利用环介导等温扩增实时荧光检测系统建立结核分枝杆菌的环介导等温扩增检测方法，同时对内外引物摩尔浓度比例、反应温度进行优化得到最佳反应条件，结果通过环介导等温扩增实时荧光曲线进行分析。通过分析环介导等温扩增实时荧光曲线可知，整个反应用时1h，大多数反应在30min内开始扩增。同时，对10倍梯度稀释的结核分枝杆菌DNA模板进行检测，环介导等温扩增实时荧光检测方法检测限可达2.4fg/反应，比PCR高100倍，对其他常见病原菌的DNA无检出，灵敏度和特异性均较好，具有广阔的临床应用前景。 第三部分：结核分枝杆菌反向斑点杂交检测方法的建立 针对结核分枝杆菌特有的插入序列IS6110，自行设计并合成生物素标记引物及特异性寡核苷酸探针。以结核分枝杆菌DNA为模板，通过PCR扩增，制备生物素标记的目的片段，产物变性与固定在带正电荷的尼龙膜上的特异性寡核苷酸探针在65℃进行杂交，每次杂交反应设有阳性对照及阴性对照。通过对探针浓度、链酶亲和素标记碱性磷酸酶的稀释倍数、杂交时间、酶促反应时间四方面进行优化得到反向斑点杂交的最优反应条件，结果通过对膜显色后是否出现蓝紫色斑点来判定阳性或者阴性。本研究建立的反向斑点杂交的特异性好，与其他常见病原菌无交叉反应，对10倍梯度稀释的结核分枝杆菌DNA模板进行检测可达到24fg/反应的检测限。与PCR相比，该方法的检测限提高了10倍，可实现结核分枝杆菌与其他常见病原菌的鉴别诊断。 第四部分：环介导等温扩增实时荧光检测方法与反向斑点杂交方法在临床标本中的应用 以临床上确诊为结核病患者的痰标本的DNA为模板，分别进行环节导等温扩增实时荧光检测和反向斑点杂交检测。所有痰标本均出现荧光扩增曲线并出现蓝紫色斑点，该结果表明本研究建立的环介导等温扩增实时荧光检测结合反向斑点杂交方法可成功的检测出结核分枝杆菌，该方法灵敏、特异，不需要精密仪器，适合在临床实验室推广使用。
[Abstract]:Tuberculosis is one of the infectious diseases that seriously threaten human health. It is one of the key prevention and control diseases in our country. It is also a public health and social problem that is the key concern of the world. According to the WHO (WHO), about 8 million 600 thousand people worldwide are now suffering from tuberculosis and 1 million 300 thousand people die from tuberculosis. The incidence of tuberculosis is increased by an average of 0.4% per year. Therefore, it is urgent to find a rapid and accurate detection method for Mycobacterium tuberculosis. In recent years, the ring mediated isothermal amplification technology has the advantages of fast, high sensitivity and strong specificity, and the reverse dot blot hybridization technique combines two techniques of gene amplification and molecular hybridization, which can be accurately and intuitively observed. It is easy to operate. This study has established a ring mediated isothermal amplification technique of Mycobacterium tuberculosis and reverse dot blot hybridization technology, which can meet the needs of rapid detection in the field and provide technical support for the prevention and control of tuberculosis.
This study is divided into four parts:
Part one: cloning and sequence analysis of Mycobacterium tuberculosis insertion sequence IS6110 gene
The specific insertion sequence IS6110 fragment of Mycobacterium tuberculosis was selected as the target sequence. A pair of specific primers were designed according to the IS6110 sequence of Mycobacterium tuberculosis published on the GenBank (GenBank login number: X17348.1). The target fragment of the PCR amplification was connected with the pGEM-T vector, and the connection product was transformed to the Escherichia coli feeling state fine. The cloned plasmid pGEM-T-IS6110. was screened by blue spot and identified by PCR and sequencing. The results showed that the IS6110 gene of Mycobacterium tuberculosis was obtained.
The second part: the establishment of loop mediated isothermal amplification real-time fluorescence detection for Mycobacterium tuberculosis.
In view of the specific insertion sequence IS6110 of Mycobacterium tuberculosis, a ring mediated isothermal amplification primer group (including one external primer and one pair of primers) was designed and synthesized by ourselves. The primers in the primer group were prepared according to a certain molar concentration to a primer group solution, and the ring mediated isothermal amplification was used for real-time fluorescence detection. The system established the method of isothermal amplification detection of Mycobacterium tuberculosis by loop mediated isothermal amplification. At the same time, the optimum reaction conditions were obtained by optimizing the molar concentration ratio of the internal and external primers and the reaction temperature. The results were analyzed by the loop mediated isothermal amplification of real time fluorescence curve. 1H, most of the reactions began to expand in 30min. At the same time, the 10 times gradient dilution of Mycobacterium tuberculosis DNA template was detected. The detection limit of the ring mediated isothermal amplification real-time fluorescence detection method was 100 times higher than PCR, and the sensitivity and specificity were better for other common pathogenic bacteria, and had a broad clinical application. Prospects.
The third part: establishment of reverse dot blot hybridization method for detection of Mycobacterium tuberculosis.
Biotin marker primers and specific oligonucleotide probes were designed and synthesized for the specific insertion sequence of Mycobacterium tuberculosis (IS6110). The target fragment of biotin was prepared by PCR amplification with DNA as a template. The specific oligonucleotide probes of product denaturation and immobilization on the nylon membrane with positive charge were 65 The optimum reaction conditions of the reverse dot blot hybridization were obtained by four aspects of the concentration of the probe, the dilution times of the chain enzyme avidin labeled alkaline phosphatase, the time of hybridization and the reaction time of the enzyme. It was positive or negative. The specificity of the reverse dot blot established in this study was good, and there was no cross reaction with other common pathogens. The detection limit of DNA template for Mycobacterium tuberculosis with 10 times gradient dilution could reach the detection limit of 24fg/ reaction. Compared with PCR, the detection limit of this method was 10 times higher than that of the method, which could realize Mycobacterium tuberculosis and others. Differential diagnosis of common pathogenic bacteria.
The fourth part: the application of loop mediated isothermal amplification real-time fluorescence detection and reverse dot blot hybridization in clinical specimens.
The DNA of sputum specimens of patients diagnosed with tuberculosis was used as a template. The real-time fluorescence detection and reverse dot blot detection were carried out respectively. All sputum specimens showed fluorescence amplification curve and blue purple spots. The results showed that the ring mediated isothermal amplification real-time fluorescence detection combined with reverse spot was established in this study. Dot blot can successfully detect Mycobacterium tuberculosis. The method is sensitive and specific, and does not require precision instruments. It is suitable for clinical laboratories.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R52;R440
本文编号：2137896
[Abstract]:Tuberculosis is one of the infectious diseases that seriously threaten human health. It is one of the key prevention and control diseases in our country. It is also a public health and social problem that is the key concern of the world. According to the WHO (WHO), about 8 million 600 thousand people worldwide are now suffering from tuberculosis and 1 million 300 thousand people die from tuberculosis. The incidence of tuberculosis is increased by an average of 0.4% per year. Therefore, it is urgent to find a rapid and accurate detection method for Mycobacterium tuberculosis. In recent years, the ring mediated isothermal amplification technology has the advantages of fast, high sensitivity and strong specificity, and the reverse dot blot hybridization technique combines two techniques of gene amplification and molecular hybridization, which can be accurately and intuitively observed. It is easy to operate. This study has established a ring mediated isothermal amplification technique of Mycobacterium tuberculosis and reverse dot blot hybridization technology, which can meet the needs of rapid detection in the field and provide technical support for the prevention and control of tuberculosis.
This study is divided into four parts:
Part one: cloning and sequence analysis of Mycobacterium tuberculosis insertion sequence IS6110 gene
The specific insertion sequence IS6110 fragment of Mycobacterium tuberculosis was selected as the target sequence. A pair of specific primers were designed according to the IS6110 sequence of Mycobacterium tuberculosis published on the GenBank (GenBank login number: X17348.1). The target fragment of the PCR amplification was connected with the pGEM-T vector, and the connection product was transformed to the Escherichia coli feeling state fine. The cloned plasmid pGEM-T-IS6110. was screened by blue spot and identified by PCR and sequencing. The results showed that the IS6110 gene of Mycobacterium tuberculosis was obtained.
The second part: the establishment of loop mediated isothermal amplification real-time fluorescence detection for Mycobacterium tuberculosis.
In view of the specific insertion sequence IS6110 of Mycobacterium tuberculosis, a ring mediated isothermal amplification primer group (including one external primer and one pair of primers) was designed and synthesized by ourselves. The primers in the primer group were prepared according to a certain molar concentration to a primer group solution, and the ring mediated isothermal amplification was used for real-time fluorescence detection. The system established the method of isothermal amplification detection of Mycobacterium tuberculosis by loop mediated isothermal amplification. At the same time, the optimum reaction conditions were obtained by optimizing the molar concentration ratio of the internal and external primers and the reaction temperature. The results were analyzed by the loop mediated isothermal amplification of real time fluorescence curve. 1H, most of the reactions began to expand in 30min. At the same time, the 10 times gradient dilution of Mycobacterium tuberculosis DNA template was detected. The detection limit of the ring mediated isothermal amplification real-time fluorescence detection method was 100 times higher than PCR, and the sensitivity and specificity were better for other common pathogenic bacteria, and had a broad clinical application. Prospects.
The third part: establishment of reverse dot blot hybridization method for detection of Mycobacterium tuberculosis.
Biotin marker primers and specific oligonucleotide probes were designed and synthesized for the specific insertion sequence of Mycobacterium tuberculosis (IS6110). The target fragment of biotin was prepared by PCR amplification with DNA as a template. The specific oligonucleotide probes of product denaturation and immobilization on the nylon membrane with positive charge were 65 The optimum reaction conditions of the reverse dot blot hybridization were obtained by four aspects of the concentration of the probe, the dilution times of the chain enzyme avidin labeled alkaline phosphatase, the time of hybridization and the reaction time of the enzyme. It was positive or negative. The specificity of the reverse dot blot established in this study was good, and there was no cross reaction with other common pathogens. The detection limit of DNA template for Mycobacterium tuberculosis with 10 times gradient dilution could reach the detection limit of 24fg/ reaction. Compared with PCR, the detection limit of this method was 10 times higher than that of the method, which could realize Mycobacterium tuberculosis and others. Differential diagnosis of common pathogenic bacteria.
The fourth part: the application of loop mediated isothermal amplification real-time fluorescence detection and reverse dot blot hybridization in clinical specimens.
The DNA of sputum specimens of patients diagnosed with tuberculosis was used as a template. The real-time fluorescence detection and reverse dot blot detection were carried out respectively. All sputum specimens showed fluorescence amplification curve and blue purple spots. The results showed that the ring mediated isothermal amplification real-time fluorescence detection combined with reverse spot was established in this study. Dot blot can successfully detect Mycobacterium tuberculosis. The method is sensitive and specific, and does not require precision instruments. It is suitable for clinical laboratories.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R52;R440
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