【作者】 张利；

【导师】 莫内； 崔步云；

【作者基本信息】 内蒙古农业大学 ， 临床兽医学， 2012， 硕士

【摘要】 为快速、准确、：便捷的检测和鉴定布鲁菌,本研究依据荧光定量PCR技术和环介导等温扩增技术(LAMP)的原理分别设计引物,建立了两种布鲁菌的检测方法并对其进行了评价。荧光定量PCR方法依据布鲁菌保守基因BCSP31设计探针和引物,LAMP方法创新性的运用布鲁菌多拷贝的插入序列IS711设计引物。试验用构建的质粒标准品对体系进行优化,确定两种方法的检测下限即灵敏度；将与布鲁菌有血清学交叉反应和种源相近的菌株DNA作为对照评价了两种方法的特异性。为保证标本的阳性检出率,本研究用荧光定量PCR技术分别对布鲁菌病患者的全血和血清中布鲁菌DNA进行了检测,这也是国内首次将血清标本用于布鲁菌的分子生物学方法检测。试验结果显示,血清标本比全血更适合作为布鲁菌的分子生物学检测用标本。通过比较,荧光定量PCR和LAMP技术均表现出较高的特异性,灵敏度都在102copy/uL的数量级水平上。在对50株布鲁菌地方菌株的鉴定中,荧光定量PCR、常规PCR以及LAMP方法与传统方法的鉴定结果符合率均为100%。而在临床血清标本的检测中,其阳性检出率分别为89%、62%和93%。对照菌株和血清的阴性符合率为100%。试验表明,两种方法用于布鲁菌病的临床诊断都具有较高的使用价值。综合考虑,前者因需要特殊仪器和要求较高的实验室环境因此更适用于对数据要求较高或需要对模板进行定量的研究,而后者因不需要专用的设备仪器,更适合用于布鲁菌病流调现场或基层卫生防疫。更多还原

【Abstract】 To detect and identify brucella in a rapid, accurate and convenient manner, we established and evaluated two measures--Fluorescence quantitative PCR and LAMP technology using different primers according to the principles of each method.Based on the conservative gene BCSP31of brucella, Fluorescence quantitative polymerase chain reaction (PCR) probe and primers are designed. LAMP uses a unique method to design primers through brucella inserting sequence IS711, which is a muLti-copy gene. The reaction system is optimized with the standard of plasmid and the lowest detection of two methods was tested. In order to evaluate the specificity of two methods, the bacteria and its similar species were tested through cross reaction and were amplified. To ensure the positive rate of specimen, we tested patient’s blood and serum specimen using fluorescence quantitative PCR and compared their positive rates. Serum was firstly used for brucella molecuLar biology detection nationwide. The resuLts showed that serum was more suitable for brucella molecuLar biological detection than whole blood. By comparison, both Fluorescent quantitative PCR and LAMP had high specificity and sensitivity. The lowest detection was within102copy/uL magnitude. In identifying50brucella strains from different places, data showed that the Fluorescence quantitative PCR, the conventional PCR and LAMP are in100%accordance with the traditional methods of brucella identification. Whereas in the test of clinical serum samples, the positive detection rates of these three methods were89%,62%and93%. The coincidence rate in the negative control was100%.Two methods are highly applicable in clinical diagnosis for brucellosis. However, the Fluorescence quantitative PCR has a higher requirement of special equipment and laboratory environment, thus more suitable for research institutes. The LAMP is more suitable for brucellosis epidemiological investigation and primary prevention given lower requirement of special equipment.更多还原