伯氏疏螺旋体分型多重实时荧光定量PCR检测方法的建立与初步应用

发布时间：2018-04-22 22:27

本文选题：伯氏疏螺旋体 + 伽氏疏螺旋体　；参考：《承德医学院》2015年硕士论文

【摘要】：莱姆病又称莱姆包柔体病或莱姆疏螺旋体病,是因感染伯氏疏螺旋体引起的由蜱为媒介传播的人畜共患传染病。伯氏疏螺旋体是莱姆病的主要病原体,在分类学上属于螺旋体目、螺旋体科中的包柔螺旋体属,也称疏螺旋体属。莱姆病最初于1977年在美国康涅狄格州的莱姆镇发现,因而得名。莱姆病临床表现复杂,早期以慢性游走性红斑为特征性表现,后可累及心脏、关节、神经系统,严重者可致死亡。莱姆病在世界上分布广泛,遍布五大洲70多个国家,且发病人数在逐年增加,疫区不断扩大,以美国发病率最高,我国也证实存在莱姆病的发病和流行,部分省(市)存在自然疫源地,严重危害人民健康和经济发展,1992年WHO已将此病列为重点防治对象。随着我国与世界各国贸易往来的与日俱增,出入境流动人口的逐年增加,为病原体的传播和转移提供了可能性,因此加强国境口岸的病原学检测成为当务之急,必须做到早发现,早防控,保障边境地区的安全与稳定。目前,莱姆病的实验室检查方法主要有血象分析、病原体分离、血清学和分子生物学方法,各有不足。病原体分离是金标准,以病变周围皮肤取样阳性率最高,但耗时耗力,不是首选方法。免疫学检测法主要有间接免疫荧光试验和酶联免疫吸附试验,快速简便,但存在一定的假阳性和假阴性。分子生物学方法主要包括常规PCR方法和实时荧光定量PCR方法,由于实时荧光定量PCR具有快速、敏感性高、特异性高、可定量、易于推广的优点,本研究拟建立能检测伯氏疏螺旋体的多重实时荧光定量PCR方法并同时对菌株的基因型分型,用于国境口岸莱姆病螺旋体的快速筛查。目的:本研究拟开展蜱媒传染病莱姆病的监测检测技术研究,建立一种伯氏疏螺旋体的多重实时荧光定量PCR的快速检测方法,可对伯氏疏螺旋体三种致病基因型伽氏疏螺旋体、阿氏疏螺旋体和狭义伯氏疏螺旋体检测并同时分型。方法:从genbank中检索伽氏疏螺旋体、阿氏疏螺旋体和狭义伯氏疏螺旋体的代表株的外膜蛋白ospc的全长序列,用mega5.0软件比对,用primerpremier5.0软件设计常规pcr全长引物,以购自美国模式菌种保藏中心的三型标准菌株的基因组核酸为模板做pcr用于ta克隆构建标准质粒,用作荧光定量pcr的质粒模板标准品。在标准质粒的保守序列设计荧光pcr的型通用引物和型特异探针,三条探针分别标记fam、texasred和cy5荧光报告基团,以标准质粒为模板,分别建立及优化伽氏疏螺旋体、阿氏疏螺旋体和狭义伯氏疏螺旋体的单重实时荧光定量pcr方法及分型多重实时荧光定量pcr方法,优化反应体系,分析其敏感性和特异性,再以三型标准菌株的基因组核酸为模板做检测以验证本方法的科学性和有效性,并初步应用于长白口岸蜱虫的莱姆病螺旋体的检测。结果:①建立的伽氏疏螺旋体单重实时荧光定量pcr方法具有很好的灵敏性,最低检测值为40copies/μl。②建立的阿氏疏螺旋体单重实时荧光定量pcr方法具有很好的灵敏性,最低检测值为5.17copies/μl。③建立的狭义伯氏疏螺旋体单重实时荧光定量pcr方法具有很好的敏感性,最低检测值为3.78copies/μl。④建立的伽氏疏螺旋体、阿氏疏螺旋体和狭义伯氏疏螺旋体的分型多重实时荧光定量pcr方法具有很好的灵敏性和特异性,对伽氏疏螺旋体、阿氏疏螺旋体和狭义伯氏疏螺旋体的最低检测值分别为40copies/μl、5.17copies/μl和38copies/μl;与蜱传病原立克次体、土拉弗朗西斯菌均无交叉反应,特异性佳。用本实验所建立的伯氏疏螺旋体taqman探针分型多重实时荧光定量pcr方法检测2009年5月采自长白山口岸的蜱虫标本72只,结果9只长角血蜱检测为阳性,均为b.garinii型,经常规pcr测序比对证实均为伽氏疏螺旋体型,结果一致,证明了本方法的可靠性。结论:本研究建立的伯氏疏螺旋体分型多重实时荧光定量PCR方法能快速检测伯氏疏螺旋体并同时分型,适用于国境口岸莱姆病螺旋体的快速检测。
[Abstract]:Lyme disease, also known as Lyme Bauer somatic disease or leim Treponema, is a zoonotic disease transmitted by a tick caused by Borrelia burgdorferi. Borrelia burgdorferi is the main pathogen of Lyme disease. It belongs to the order of helix taxonomy, the genus Helix in the family spironifolia, also known as sparsely. Lyme disease is the most common disease. First found in Lyme Town, Connecticut, in 1977, it was named. Lyme disease was characterized by complicated clinical manifestations and characterized by chronic walking erythema, which could involve heart, joint, nervous system and death. Lyme disease was widely distributed around the world in more than 70 countries and increased year by year in five continents. In addition, the epidemic area is expanding and the incidence of the disease is the highest in the United States. China has also confirmed the incidence and prevalence of Lyme disease. Some provinces (cities) have natural foci, seriously endangering the people's health and economic development. In 1992, WHO has listed the disease as the key control object. The increase year by year provides the possibility for the transmission and transfer of pathogens. Therefore, it is urgent to strengthen the detection of etiology at frontier ports. It is necessary to detect early, prevent and control early and ensure the safety and stability of the border areas. The pathogen separation is the gold standard, the positive rate of the skin sampling around the lesion is the highest, but the time consuming force is not the first choice. The immunological detection method is mainly indirect immunofluorescence test and enzyme linked immunosorbent test, fast and simple, but there is a certain false positive and false negative. The molecular biological methods mainly include the conventional PC. R method and real-time fluorescence quantitative PCR method, because real-time fluorescence quantitative PCR has the advantages of rapid, high sensitivity, high specificity, quantitative and easy to be popularized. This study intends to establish a multiple real-time quantitative PCR method for detection of burgospira burgdorferi and the genotyping of the strain, which is used for the rapid growth of Lyme disease spiral body at the frontier port. Objective: to develop a monitoring and detection technique for Lyme disease of tick borne diseases, and to establish a rapid detection method for multiple real-time quantitative PCR of Borrelia burgdorferi, which can be used to detect and classify three kinds of pathogenic gal spirulae, heliospira alsosii and Borrelia burgdorferi. Methods: to retrieve the full length of the outer membrane protein ospC of galleto helix, alsoprin and the representative strain of Borrelia narrow Borrelia from GenBank, and use the mega5.0 software to design the conventional PCR full-length primers with primerpremier5.0 software. PCR was used to construct standard plasmids for TA cloning and used as a standard plasmid template for fluorescent quantitative PCR. The universal primers and type specific probes of fluorescent PCR were designed in the conservative sequence of standard plasmids. Three probes were labeled fam, texasred and Cy5 fluorescent groups respectively, and the standard plasmid was used as a template to establish and optimize the galeron sparsely, respectively. The single real time fluorescence quantitative PCR method and the multiple real-time fluorescence quantitative PCR method were used to optimize the reaction system and analyze its sensitivity and specificity. Then the genomic DNA of the three standard strain was used as a template to verify the scientificity and effectiveness of the method, and it was preliminarily applied to the long white. The detection of Lyme disease helix of ticks of port ticks. Results: (1) the method of single weight real time quantitative PCR for galleto Treponema has good sensitivity. The minimum detection value is 40copies/ Mu L., the single weight real-time fluorescence quantitative PCR method has good sensitivity, and the minimum detection value is 5.17copies/ Mu L. 3. The single weight real-time fluorescence quantitative PCR method with narrow sense Borrelia has very good sensitivity. The minimum detection value is the galeri spiral body established by 3.78copies/ Mu L.. The multiple real-time fluorescence quantitative PCR method of the alsotrea and narrow sense Borrelia has good sensitivity and specificity. The lowest detection values of Borrelia and Borrelia narrow sense were 40copies/, 5.17copies/, l and 38copies/ Mu respectively. No cross reaction was found with the tick borne Rickettsia, Tur La Francis bacteria had no cross reaction, and the specificity was good. The multiple real-time fluorescent quantitative PCR method based on the bersate Borrelia TaqMan probe established in this experiment was used for the detection of 2009. In May, 72 ticks collected from Changbai Mountain port were collected from the Changbai Mountain port. The results showed that 9 chlorpyrifos were positive, all of them were type b.garinii. The results of regular PCR sequencing comparison were all galeri spiral type. The results were consistent and proved the reliability of this method. Borrelia burgdorferi is also suitable for rapid detection of Borrelia burgdorferi at frontier ports.

【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R440

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