环介导等温扩增（LAMP）检测新疆北疆地区布鲁氏菌病原的方法研究

发布时间：2018-06-17 03:38

本文选题：布鲁氏菌 + MLVA-16　；参考：《石河子大学》2014年硕士论文

【摘要】：目的：布鲁氏菌病在全球范围内都是一个亟待解决的难题，每年因布鲁氏菌病给许多国家和地区的畜牧业带来巨大的经济损失，同时对人类的健康形成了极大的威胁。每年报告的人患布鲁氏菌病就超过50万例。研究表明，布鲁氏菌的致病能力主要依靠其在宿主中的生存和繁殖能力，并没有公认的一般致病细菌具有的毒力因子。布鲁氏菌致病的因子主要依靠LPS、OMP和其他的毒力相关因子。OMP25是布鲁氏菌的一种外膜蛋白，而omp25基因在不同种布鲁氏菌之间具有高度的同源性。环介导等温扩增（LAMP）是一种在近年来有极大发展的核酸扩增技术，利用对靶基因的6个区域，设计4条引物，，在65℃恒温条件下进行扩增，40-60min结束反应，通过颜色就可方便的判定反应结果。LAMP具有便捷和特异性高的特性，对于能够广泛应用于检测家畜间的布鲁氏菌病具有十分重要的意义。方法：首先，调查了新疆北疆地区布鲁氏菌病的流行情况。从新疆乌苏、阿勒泰、玛纳斯三个地区和西部牧业采集家畜血液样品共1721份进行琥红平板和试管凝集试验检测，其中羊血清1053份、牛血清668份。同时对新疆上述四个地方的母畜流产胎儿内容物进行布鲁氏菌病原分离、鉴定并用MLVA-16方法进行分型鉴定。其次，为建立一种快速、高效、准确诊断和检测布鲁氏菌的LAMP检测技术的方法，针对布鲁氏菌外膜蛋白基因Omp25区域设计6条引物，65℃反应60min，85℃5min终止反应后，用SYBR Green I染料做指示剂，根据颜色判定结果。为了评价建立的LAMP方法的特异性和敏感性，取乌苏、阿勒泰、玛纳斯三个地区和西部牧业患病动物体内血液分离布鲁氏菌并进行培养，用LAMP和PCR方法进行平行检测。结果：阳性率都比较高，最高达13.8%，羊感染率平均为11.1%，牛感染率平均为4.04%。表明了布鲁氏菌病在这些地区的发生率比较高。分离株用常规分型方法鉴定为羊种3型，MLVA-16方法鉴定该菌株与德国分离株Bruce0261关系最近。LAMP方法最低检测出的灵敏度为4.2fg/μL布鲁氏菌基因组DNA。本方法特异性比较好，琼脂糖凝胶电泳检测布鲁氏菌管显示阳性，并且都有特征性的梯形条带，而大肠杆菌、卡介苗、金黄色葡萄球菌、李氏杆菌都没有出现扩增现象。样品分离菌LAMP和PCR符合率100%，并且LAMP的灵敏度要高于PCR。选取的乌苏、阿勒泰、玛纳斯三个地区和西部牧业的血样培养的布鲁氏菌，用LAMP和PCR检测符合率100% 结论：（1）在新疆省内乌苏、阿勒泰、玛纳斯三个地区和西部牧业血清学实验检测结果表明这些地区的布鲁氏菌病的阳性率较前些年有上升趋势。MLVA-16实验结果表明羊种布鲁氏菌3型，基因型42型为北疆地区主要流行基因型。（2）本研究选取了布鲁氏菌共有基因omp25，建立了布鲁氏菌环介导等温扩增检测方法，实验表明LAMP具有高敏感性和特异性，并用LAMP方法鉴定了从新疆乌苏、阿勒泰、玛纳斯三个地区和西部牧业采取的分离菌，和PCR结果进行了比较，符合率较高。该方法建立了一种快速，高效，便捷的检测布鲁氏菌的方法，为基层检测布鲁氏菌提供了一种可选的快速诊断方法。
[Abstract]:Objective: Brucellosis is an urgent problem all over the world. Every year, brucellosis has brought huge economic losses to the livestock industry in many countries and regions, and poses a great threat to human health. More than 500 thousand cases of brucellosis are reported per year. The ability of the disease mainly depends on its survival and reproductive capacity in the host, and there is no universally recognized virulence factor of the general pathogenic bacteria. The factors causing the disease of Brucella are mainly dependent on LPS, OMP and other virulence related factors.OMP25 are a kind of outer membrane protein of Brucella, and the omp25 base has a high level between different Brucella species. Homology. Ring mediated isothermal amplification (LAMP) is a kind of nucleic acid amplification technology which has been greatly developed in recent years. Using 6 regions of target gene, 4 primers are designed, amplification at the constant temperature of 65, 40-60min end reaction, and the convenient and specific characteristic of.LAMP, which can be conveniently determined by color. It is very useful for detecting brucellosis among livestock.
Methods: first, the epidemic situation of brucellosis in northern Xinjiang area was investigated. A total of 1721 blood samples collected from three regions of Xinjiang, Aletai, Manasi and Western pastoral areas were tested for succinic red plate and test tube agglutination test, including 1053 sheep sera and 668 bovine blood albumin. At the same time, there were four local animals in Xinjiang. The abortion fetal contents were separated, identified and identified by MLVA-16 method. Secondly, a rapid, efficient, accurate diagnosis and detection method of Brucella LAMP was established. 6 primers were set for the Omp25 region of the outer membrane protein gene of Brucella, 65 C reaction 60min, and 85 centigrade 5min after the termination of the reaction. In order to evaluate the specificity and sensitivity of the established LAMP method, SYBR Green I dyestuff was used as an indicator to determine the specificity and sensitivity of the established LAMP method. The blood separation of Brucella from three regions of Wusu, Aletai, Manasi and Western Pastoral animals was isolated and cultured, and the LAMP and PCR methods were used to carry out parallel detection.
Results: the positive rate was high, the highest was 13.8%, the average rate of sheep infection was 11.1%, the average rate of cattle infection was 4.04%., which showed that the incidence of brucellosis was higher in these areas. The isolated strain was identified as the sheep species 3 by the conventional typing method, and the MLVA-16 method identified the relationship between the strain and the German isolate Bruce0261 recently by the.LAMP method. The sensitivity of the method was 4.2fg/ mu L Brucella genomic DNA., the specificity of this method was better. Agarose gel electrophoresis detected brucellosis tube positive, and there were characteristic trapezoid bands, while Escherichia coli, Bacillus Calmette vaccine, Staphylococcus aureus and Listeria had no amplification phenomenon. The coincidence rate of sample isolates LAMP and PCR was 10 0%, and the sensitivity of LAMP is higher than that of Wusu, which is selected by PCR., Aletai, three regions of Manasi and the blood samples from the western animal husbandry, and the coincidence rate of LAMP and PCR is 100%.
Conclusions: (1) the test results of the serological tests in three regions and western regions of Xinjiang province in Xinjiang province showed that the positive rate of brucellosis in these areas was higher than that of the previous years. The results of the.MLVA-16 experiment showed that the sheep Brucella type 3 and genotype 42 were the main genotypes in Northern Xinjiang. (2) the study was selected. The common gene omp25 of Brucella was taken, and the method of isothermal amplification of Brucella was established. The experiment showed that LAMP had Gao Min sensibility and specificity, and the isolated bacteria from three regions of Wusu, Aletai, Manasi, Xinjiang and Western pasture were identified by LAMP method. The results were compared with the results of PCR, and the coincidence rate was high.
This method has established a fast, efficient and convenient method for Brucella detection, providing an alternative rapid diagnostic method for Brucella.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R516.7

【相似文献】