臺灣博碩士論文加值系統

草綠色鏈球菌（viridans group streptococci）為人體口腔內之正常菌叢（normal flora），但卻也是齲齒（dental caries）、感染性心內膜炎（infective endocarditis）、敗血症（septicemia）及許多深部組織嚴重感染之重要病原菌。草綠色鏈球菌至少有20種以上的菌種（species），目前分為5群：S. mutans group, S. mitis group, S. milleri group, S. salivarius group和S. bovis group。沒有單一系統可以來分類草綠色鏈球菌，一般依照菌落生長特徵（growth characteristics）、在血液培養基的溶血型式、細胞壁抗原組成（antigenic composition）、生化反應（biochemical reactions）和最近發展的分子生物學方法來分類。
60 kDa的熱刺激蛋白質基因（groEL基因）普遍存在各細菌中，近年來有報告以此基因當工具，用來作葡萄球菌（staphylococci）或分枝桿菌（mycobacteria）各菌種間的鑑定。本研究已找到S. gordonii的groESL基因，它包含282 bp的groES和1623 bp的groEL，GroEL（groEL）分別和S. pneumoniae、E. coli有91%(81%)、60%(62%)的相似性；而GroES（groES）分別有73%(71%)、39%(45%)的相似性。分析此段基因序列，發現與其它革蘭氏陽性細菌類似，在啟動子（promoter）區域有調控基因序列-CIRCE，而在groEL基因後面有長的反重覆序列（inverted repeat, IR），可能為基因轉錄停止訊號（rho-independent transcriptional terminator）。在groESL 5''端及3''端分別發現2個可能的基因，經比對，1個類似ATP-binding cassette (ABC)基因位於groES基因5''端；另1個為未知基因（an unknown gene），位於groEL基因3''端。
根據S. gordonii groESL基因高度相似的序列，設計出degenerate PCR引子，也將S. oralis的groESL基因全長定序並得到其它草綠色鏈球菌的片段groEL基因序列。其中S. oralis的GroEL（groEL）和S. gordonii、S. pneumoniae、E. coli有93% (82%)、98% (91%)及60% (62%)的相似性。而實驗中所有草綠色鏈球菌的片段groEL基因序列兩兩相比，皆至少有75%以上的DNA相似性和85%以上的胺基酸相似性。
以groEL基因為標的，分別利用點漬雜交法（dot blot hybridization）、直接定序法（direct sequencing）、和PCR-RFLP分析來嘗試鑑定草綠色鏈球菌。初步結果認為上述3種方法都可行，但其臨床上之實際應用需要再繼續評估，以設計敏感度、特異度均高，且簡單易操作的方法。

The oral or viridans group streptococci form a significant part of the normal flora of the human oral cavity and are associated with several disease conditions including dental caries, infective endocarditis and septicemia as well as purulent infections. Currently the viridans group streptococci taxonomically could be divided into five major clusters which included at least 20 species. These are S. mutans group, S. mitis group, S. milleri group, S. salivarius group and S. bovis group. No single system of classification suffices for the identification of this heterogeneous of organisms. Instead, classification depends on a combination of features including pattern of hemolysis observed on blood agar plates, antigenic composition, growth characteristics, biochemical reactions, and more recently, genetic analysis.
The groEL gene, which encodes 60-kDa heat shock protein (GroEL), is ubiquitous and highly conserved among bacteria. It has been recently reported by using groEL gene as an alternative target for species-specific identification of staphylococci or mycobacteria. In this study, the S. gordonii groESL operon containing groES (282 bp) and groEL (1623 bp) was cloned and sequenced. The GroEL (groEL) of S. gordonii had 91% (81%) homology to S. pneumoniae and 60% (62%) to E. coli while GroES (groES) had 73% (71%) homology to S. pneumoniae and 39% (45%) to E. coli. Like other Gram-positive bacteria, a putative transcriptional promoter upstream of groES that was comprised of -35 and -10 hexamers franked downstream by the conserved Gram-positive heat shock gene regulatory sequence, CIRCE. A large inverted repeat that may function as a rho-independent transcriptional terminator was located downstream of groEL gene. In addition, the gene encoded ATP-binding cassette (ABC) transporter protein located upstream of groES and another gene located downstream of groEL were found.
Degenerate PCR primers derived from conserved regions of the groESL operon of S. gordonii were used to amplify groESL of other species of viridans group streptococci. Sequences were determined for entire groESL of S. oralis and partial groEL fragment of the other species. The GroEL (groEL) homology of S. orals with S. gordonii, S. pneumoniae and E. coli were 93% (82%), 98% (91%) and 60% (62%), respectively. Comparing the sequence of GroEL and groEL among viridans group streptococci in this study, there were 75% and 85% homology among them. For application of species identification, the dot blot hybridization, direct sequencing and PCR-RFLP were used. Our results demonstrate that groEL-based identification method has the potential to be an alternative method for identification of the viridans group streptococci. Further work is required to test more strains of bacteria for designing a simple performing method with both high specificity and sensitivity.

英文摘要……………………………………………1
中文摘要……………………………………………4
緒論…………………………………………………6
實驗設計與目的……………………………………12
材料與方法…………………………………………13
結果…………………………………………………37
討論…………………………………………………45
實驗結果之附圖表…………………………………58
參考文獻……………………………………………85

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