本文選題：伯氏疏螺旋體 + 伽氏疏螺旋體�。� 參考：《承德醫(yī)學院》2015年碩士論文

【摘要】：萊姆病又稱萊姆包柔體病或萊姆疏螺旋體病,是因感染伯氏疏螺旋體引起的由蜱為媒介傳播的人畜共患傳染病。伯氏疏螺旋體是萊姆病的主要病原體,在分類學上屬于螺旋體目、螺旋體科中的包柔螺旋體屬,也稱疏螺旋體屬。萊姆病最初于1977年在美國康涅狄格州的萊姆鎮(zhèn)發(fā)現(xiàn),因而得名。萊姆病臨床表現(xiàn)復雜,早期以慢性游走性紅斑為特征性表現(xiàn),后可累及心臟、關節(jié)、神經系統(tǒng),嚴重者可致死亡。萊姆病在世界上分布廣泛,遍布五大洲70多個國家,且發(fā)病人數(shù)在逐年增加,疫區(qū)不斷擴大,以美國發(fā)病率最高,我國也證實存在萊姆病的發(fā)病和流行,部分省(市)存在自然疫源地,嚴重危害人民健康和經濟發(fā)展,1992年WHO已將此病列為重點防治對象。隨著我國與世界各國貿易往來的與日俱增,出入境流動人口的逐年增加,為病原體的傳播和轉移提供了可能性,因此加強國境口岸的病原學檢測成為當務之急,必須做到早發(fā)現(xiàn),早防控,保障邊境地區(qū)的安全與穩(wěn)定。目前,萊姆病的實驗室檢查方法主要有血象分析、病原體分離、血清學和分子生物學方法,各有不足。病原體分離是金標準,以病變周圍皮膚取樣陽性率最高,但耗時耗力,不是首選方法。免疫學檢測法主要有間接免疫熒光試驗和酶聯(lián)免疫吸附試驗,快速簡便,但存在一定的假陽性和假陰性。分子生物學方法主要包括常規(guī)PCR方法和實時熒光定量PCR方法,由于實時熒光定量PCR具有快速、敏感性高、特異性高、可定量、易于推廣的優(yōu)點,本研究擬建立能檢測伯氏疏螺旋體的多重實時熒光定量PCR方法并同時對菌株的基因型分型,用于國境口岸萊姆病螺旋體的快速篩查。目的:本研究擬開展蜱媒傳染病萊姆病的監(jiān)測檢測技術研究,建立一種伯氏疏螺旋體的多重實時熒光定量PCR的快速檢測方法,可對伯氏疏螺旋體三種致病基因型伽氏疏螺旋體、阿氏疏螺旋體和狹義伯氏疏螺旋體檢測并同時分型。方法:從genbank中檢索伽氏疏螺旋體、阿氏疏螺旋體和狹義伯氏疏螺旋體的代表株的外膜蛋白ospc的全長序列,用mega5.0軟件比對,用primerpremier5.0軟件設計常規(guī)pcr全長引物,以購自美國模式菌種保藏中心的三型標準菌株的基因組核酸為模板做pcr用于ta克隆構建標準質粒,用作熒光定量pcr的質粒模板標準品。在標準質粒的保守序列設計熒光pcr的型通用引物和型特異探針,三條探針分別標記fam、texasred和cy5熒光報告基團,以標準質粒為模板,分別建立及優(yōu)化伽氏疏螺旋體、阿氏疏螺旋體和狹義伯氏疏螺旋體的單重實時熒光定量pcr方法及分型多重實時熒光定量pcr方法,優(yōu)化反應體系,分析其敏感性和特異性,再以三型標準菌株的基因組核酸為模板做檢測以驗證本方法的科學性和有效性,并初步應用于長白口岸蜱蟲的萊姆病螺旋體的檢測。結果:①建立的伽氏疏螺旋體單重實時熒光定量pcr方法具有很好的靈敏性,最低檢測值為40copies/μl。②建立的阿氏疏螺旋體單重實時熒光定量pcr方法具有很好的靈敏性,最低檢測值為5.17copies/μl。③建立的狹義伯氏疏螺旋體單重實時熒光定量pcr方法具有很好的敏感性,最低檢測值為3.78copies/μl。④建立的伽氏疏螺旋體、阿氏疏螺旋體和狹義伯氏疏螺旋體的分型多重實時熒光定量pcr方法具有很好的靈敏性和特異性,對伽氏疏螺旋體、阿氏疏螺旋體和狹義伯氏疏螺旋體的最低檢測值分別為40copies/μl、5.17copies/μl和38copies/μl;與蜱傳病原立克次體、土拉弗朗西斯菌均無交叉反應,特異性佳。用本實驗所建立的伯氏疏螺旋體taqman探針分型多重實時熒光定量pcr方法檢測2009年5月采自長白山口岸的蜱蟲標本72只,結果9只長角血蜱檢測為陽性,均為b.garinii型,經常規(guī)pcr測序比對證實均為伽氏疏螺旋體型,結果一致,證明了本方法的可靠性。結論:本研究建立的伯氏疏螺旋體分型多重實時熒光定量PCR方法能快速檢測伯氏疏螺旋體并同時分型,適用于國境口岸萊姆病螺旋體的快速檢測。
[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.

【學位授予單位】：承德醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R440

【參考文獻】

相關期刊論文 前1條

1 潘正論;張源潮;鄭加田;朱凱;;重癥萊姆病1例[J];慢性病學雜志;2013年09期

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本文編號：1789227