本文關鍵詞： PFGE 巴爾通體 流行病　出處：《中國疾病預防控制中心》2008年碩士論文　論文類型：學位論文

【摘要】： 脈沖場凝膠電泳是1984年由Schwartz和Canter發(fā)明的,后經(jīng)一系列的發(fā)展,最終成為了現(xiàn)在分子生物學中的一項關鍵技術。 脈沖場凝膠電泳在細菌的研究中已普遍應用,特別是在疾病的爆發(fā)流行中,已顯示出其強大的應用價值。它不但可以單獨用于追蹤傳染源,而且可以輔助現(xiàn)場流行病學調(diào)查,為其提供菌群之間相互關系的證據(jù),現(xiàn)已成為疾病爆發(fā)中追蹤溯源必不可少的工具。最近,研究發(fā)現(xiàn)巴爾通體的各個種與所致疾病有很強的相關性,即不同種可以導致不同的疾病,而各種內(nèi)不同基因型菌株所致疾病是否有特異性,目前國內(nèi)外還沒有相關的研究。此外,對于巴爾通體在不同宿主動物中的感染情況,國外多為簡單的陽性率的報道,不能準確的反映各宿主動物感染各種巴爾通體的真實情況。在這種情況下,急需一種分型能力強、簡便、可靠、可以分辨種間種內(nèi)的分子生物學技術,以用于巴爾通體疾病的預防控制。 本研究通過對菌株濃度的選擇,限制性內(nèi)切酶的選擇,參數(shù)的優(yōu)化,傳代比較實驗四個實驗過程,建立了巴爾通體的PFGE方法。其參數(shù)分別為:菌液濃度≥4.5(比濁儀讀數(shù)),限制性內(nèi)切酶首選Smal,電泳參數(shù)為參數(shù)三(EP-C)。此外,我們在巴爾通體的傳代比較實驗中發(fā)現(xiàn),漢賽巴爾通體經(jīng)兩次傳代有20%的概率發(fā)生同條帶的丟失,而伊利莎白巴爾通體未發(fā)現(xiàn)變異。 隨后,我們通過Bionumerics數(shù)據(jù)庫軟件對52株巴爾通體進行了聚類分析(這些菌株分別來自于山東,北京,河南地區(qū)),發(fā)現(xiàn)PFGE可以用于巴爾通體的種間鑒別診斷。此外,通過對巴爾通體宿主貓和犬的流行病學分析,發(fā)現(xiàn)貓與犬感染的巴爾通體DNA分型差別大,相似度低于60%,推測可能為巴爾通體的不同種。 最后,我們通過對部分實驗菌株進行gltA基因擴增,測序,并對其進行同源性分析和進化樹分析,發(fā)現(xiàn)分離自犬和貓的巴爾通體各自聚為一類,且彼此分離沒有交叉。此外,通過NCBI的BLAST比對,所測的來自于貓的實驗菌株全部為漢賽巴爾通體,而來自于犬的實驗菌株全為文氏巴爾通體伯格霍夫亞種(B.vinsonii subsp.berhoffii)。所有這些結果與PFGE分類結果一致。 綜上所述,通過本研究我們建立了巴爾通體的PFGE方法,通過對其進行聚類分析發(fā)現(xiàn)PFGE可以用于巴爾通體的種間分類,且宿主貓,犬中感染的巴爾通體是不同種的巴爾通體。此外三地區(qū)宿主貓中,感染的巴爾通體(除M9HN)均為漢賽巴爾通體,且三地區(qū)的菌株的優(yōu)勢DNA型均不同。
[Abstract]:In 1984, pulsed field gel electrophoresis was invented by Schwartz and Canter. After a series of developments, it has become a key technology in molecular biology. Pulse field gel electrophoresis has been widely used in the study of bacteria, especially in the outbreak of disease, has shown its strong application value, it can not only be used to track the source of infection. And it can assist in the field epidemiological investigation and provide evidence of the relationship between the flora. It has become an essential tool for tracing the source of the disease outbreak. Recently. The study found that each species of Bartonella has a strong correlation with the resulting disease, that is, different species can lead to different diseases, and the disease caused by different genotypes of various strains is specific. At present, there is no related research at home and abroad. In addition, the infection of Bartonella in different host animals is mostly reported in foreign countries. It can not accurately reflect the real situation of the host animals infected with various Bartonella. In this case, the need for a strong typing ability, simple, reliable, can distinguish between species of molecular biology technology. For the prevention and control of Bartonella disease. Through the selection of strain concentration, the selection of restriction endonuclease, the optimization of parameters, the passage comparison experiment four experimental processes. The PFGE method of Baltonella was established. The parameters were as follows: the concentration of bacterial liquid 鈮，

本文編號：1491339