炭疽芽孢杆菌特征基因恒温扩增检测方法的研究

发布时间：2018-01-14 06:26

本文关键词：炭疽芽孢杆菌特征基因恒温扩增检测方法的研究　出处：《中国科学院研究生院（武汉病毒研究所）》2007年博士论文　论文类型：学位论文

【摘要】： 炭疽是由炭疽芽孢杆菌引起的严重急性传染病，同时由于炭疽芽孢在环境中的抵抗力极强，是潜在的生物战剂之一，因此研究炭疽芽孢杆菌的检测对于临床诊断炭疽和防范生物恐怖威胁具有重要的意义。针对以上需求，本研究将恒温扩增技术应用于炭疽芽孢杆菌的检测，发展了炭疽芽孢杆菌的恒温扩增检测方法，根据扩增技术的不同，分为环介导的恒温核酸扩增技术(loop-mediated isothermal amplification，LAMP)和链置换扩增技术(strand displacement amplification，SDA)两种。炭疽芽孢杆菌的LAMP检测方法选择Ba813序列、pag基因和capB基因为检测目标，分别代表炭疽芽孢杆菌的基因组和两个毒力编码质粒pXO1和pXO2。以炭疽芽孢杆菌A16纯培养物为检测对象，LAMP方法的检测灵敏度为10个芽孢，比多重PCR灵敏度高一个数量级。通过对LAMP扩增产物酶切及46株细菌的LAMP扩增试验表明该方法检测炭疽芽孢杆菌具有较高的特异性。随后以混有炭疽芽孢的粉末为模拟实际样品初步评价了LAMP方法在实际检测中的应用前景。以上试验均采用凝胶电泳方法检测LAMP扩增产物，本研究也尝试用三种不同的荧光染料EvaGreen~(TM)、SYBR~(?) Gold以及EB分别检测Ba813序列、pag基因和capB基因LAMP扩增产物，发展炭疽芽孢杆菌多荧光共检测LAMP方法，，使LAMP方法的结果判断更加简单、直观。炭疽芽孢杆菌的SDA检测方法选择pag基因为检测目标，以凝胶电泳方法检测SDA扩增产物，初步评价了该方法的灵敏度和特异性。由于SDA扩增产物在凝胶电泳检测时经常有拖尾现象，目前SDA技术的常用检测方法是荧光偏振和FRET技术，其信号检测依赖昂贵的荧光检测仪器。为此，本研究发展了一种新的SDA-ELISA检测方法，通过酶学显色使SDA产物检测更加简单直观。采用优化的反应条件，该方法检测炭疽芽孢杆菌的特异性高，检测灵敏度为10个芽孢，比对应的凝胶电泳方法高10倍。本研究发展的两种炭疽芽孢杆菌的恒温扩增检测方法具有较高的灵敏度和特异性且对仪器依赖程度低，具有良好的应用前景，该方法的发展和应用为炭疽芽孢杆菌的检测提供了新的手段。
[Abstract]:Anthrax is caused by Bacillus anthracis severe acute infectious disease, and because the resistance of Bacillus anthracis in the environment is extremely strong, is one of the potential biological warfare agents, so the detection of Bacillus anthracis has important significance for clinical diagnosis and prevention of anthrax bioterrorism threat. According to the above requirements, this study will detect the amplification the technology applied to Bacillus anthracis, Bacillus anthracis developed isothermal amplification methods, according to different amplification technology, divided into a loop mediated isothermal nucleic acid amplification technology (loop-mediated isothermal amplification, LAMP) and strand displacement amplification (strand displacement, amplification, SDA) two.
LAMP method for detection of Bacillus anthracis Ba813 sequences, PAG gene and capB gene as the target detection, representing the Bacillus anthracis genome and two virulence plasmid pXO1 encoding and pXO2. in Bacillus anthracis A16 pure culture method for object detection, LAMP detection sensitivity was 10 spores, ratio of multiple PCR sensitivity an order of magnitude higher. The LAMP products were digested and 46 strains of bacteria were amplified by LAMP test showed that the specificity for detection of Bacillus anthracis is higher. Then mixed with anthrax spores powder application to simulate the actual sample evaluation of the LAMP method in the actual testing. These tests were using gel electrophoresis method for detection of LAMP amplification products, this study tries to use three different fluorescent dye EvaGreen~ (TM), SYBR~ (?) Gold and EB were used to detect the Ba813 sequence of PAG gene and capB gene LAMP The LAMP method of multi fluorescence detection of Bacillus anthracis was developed, which made the results of LAMP method more simple and intuitionistic.
SDA method for detection of Bacillus anthracis PAG gene as the target detection, PCR products by gel electrophoresis method for detection of SDA, a preliminary evaluation of the sensitivity and specificity of this method. The SDA amplification products in gel electrophoresis often tailing phenomenon, the current SDA technology commonly used detection methods of fluorescence polarization and FRET technology, the signal detection on fluorescence detection instrument is expensive. Therefore, this study developed a new method for determination of SDA-ELISA by enzymatic colorimetric detection of SDA product to make more simple and intuitive. The optimized reaction conditions, the specificity of the method for detection of Bacillus anthracis, the detection sensitivity is 10 to 10 times higher than the spores. Gel electrophoresis method corresponding to.
The two isothermal amplification detection methods of Bacillus anthracis developed in this study have high sensitivity and specificity, and have low potential for instrument dependence, which has good application prospects. The development and application of this method provide a new way for the detection of Bacillus anthracis.

【学位授予单位】：中国科学院研究生院（武汉病毒研究所）
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R378

【引证文献】