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系統識別號 U0026-0812200915053626
論文名稱(中文) 開發鑑定臨床厭氧菌之寡核苷酸晶片
論文名稱(英文) Development of an Oligonucleotide Array for Identification of Clinically Important Anaerobic Bacteria
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 97
學期 1
出版年 98
研究生(中文) 林佑姿
研究生(英文) Yu-tzu Lin
電子信箱 t3695101@mail.ncku.edu.tw
學號 t3695101
學位類別 碩士
語文別 中文
論文頁數 84頁
口試委員 指導教授-張長泉
口試委員-吳俊忠
口試委員-柯文謙
口試委員-鄧麗珍
中文關鍵字 寡核苷酸晶片  厭氧菌 
英文關鍵字 intergenic spacer region  anaerobic bacteria  oligonucleotide array 
學科別分類
中文摘要 厭氧菌在臨床上造成許多不同的感染,有些屬嚴重感染。傳統方法鑑定厭氧菌需厭氧培養裝置且較為耗時,因此若有快速且正確的鑑定方法,在臨床上具有價值。本研究之目的在發展寡核苷酸晶片(oligonucleotide array)以鑑定臨床上較常見之厭氧菌,該晶片之原理是以核糖核酸內轉錄區(intergenic spacer region, ITS)為標的,設計寡核苷酸探針(oligonucleotide probe)點漬於尼龍膜上,將待測菌種之ITS區域以PCR放大後與尼龍上之寡核苷酸探雜合(hybridization)反應,以鑑定厭氧菌。本研究對27種厭氧菌及Veillonella屬設計了52個專一性探針,包含42個菌種特異性(species-specific)探針及6個菌群特異性(group-specific)探針,將其點製成0.9 cm × 0.8 cm的微矩陣晶片(oligonucleotide array chip)。以此寡核苷酸晶片鑑定119目標參考菌株(reference target strains),其中有4菌株以晶片鑑定結果與菌種中心種名不符合,進一步定序這4株菌之16S rRNA基因以確認其種名,結果證實晶片對其中3株菌的鑑定是正確的,另外一株為非目標菌株。再以晶片鑑定189株目標臨床分離株(以Rapid ID 32A或RapID ANA II 鑑定),被正確鑑定的有160株,其餘29株晶片和生化反應鑑定的結果不符合,這些不符合的菌株再以Rapid ID 32A及16S rRNA基因定序以確認其種名。結果證實在這189株菌中,181株為目標菌株,另外有兩株非目標臨床菌株被證實應為目標菌株。在總數183株目標臨床菌株中, 169株被晶片正確鑑定。另以105株非目標菌株(50種,包含45株參考菌株及60株臨床菌株)測試晶片的特異性,有1株被錯誤鑑定。綜合上述結果,晶片鑑定之靈敏度(sensitivity)為95.3% (287/301),特異性(specificity)為99.0% (104/105)。目前成果顯示,此晶片可成為鑑定臨床厭氧菌之有力工具。
英文摘要 Anaerobic bacteria are clinically important pathogens that can cause a wide variety of infections. Because of some serious infections are caused by anaerobic bacteria and conventional identification of these bacteria is time-consuming, rapid and accurate identification of these microorganisms may have clinical importance. In this study, an oligonucleotide array was developed to identify clinically frequently isolated anaerobes. Species-specific oligonucleotide probes were designed from the 16S-23S ribosomal DNA intergenic spacer (ITS) regions and immobilized on nylon membrane. The ITS regions of anaerobic bacteria were amplified by PCR and hybridized to probes on the membrane for species identification. A total of 52 probes, including 46 species-specific probes and 6 group-specific probes, were used to fabricate an oligonucleotide array (0.9 cm × 0.8 cm) to identify 27 species of anaerobic bacteria and the genus Veillonella. A collection of 119 reference target strains (strains I aimed to identify) were tested by the array for species identification and four strains were found to produce discrepant identification by the array. The four discordant strains were further analyzed by sequencing of the 16S rRNA gene for species clarification, three strains were found to be correctly identified by the array, with the remaining one being an nontarget strains. In addition, a collection of 189 target clinical isolates (identified by Rapid ID 32A or RapID ANA II) were identified by the array and among them, 160 stains were correctly identified, with 29 strais producing discrepant identification. These 29 discrepant clinical isolates were retested with Rapid ID 32A and sequencing of the 16S rRNA gene. The results demonstrated that among the 189 clinical isolates, 181 were target strains. In addition, two additional strains that was originally identified as nontarget strains were found to be target strains by array hybridization and sequencing of the 16S rRNA gene. Of the 183 target clinical isolates, 169 were correctly identified by the array. Moreover, 105 nontarget strains (50 species, including 45 reference strains and 60 clinical isolates) were used for specificity test of the array. Among these nontarget strains, only one strain was misidentified by the array. In conclusion, The sensitivity and specificity of the array for identification of the 27 species of anaerobic bacteria and Veillonella were 95.3% (287/301) and 99.0% (104/105), respectively. The current array can be used as an accurate alternative of conventional methods for identification of clinically important anaerobic bacteria.
論文目次 中文摘要.........................................................................................................Ⅰ
英文摘要........................................................................................................III
誌謝.................................................................................................................Ⅴ
目錄.................................................................................................................VI
表目錄........................................................................................................... Ⅷ
圖目錄.............................................................................................................IX

緒論
厭氧菌(Anaerobic bacteria)......................................................................1
治療厭氧菌常用抗生素……...................................................................4
實驗室診斷(Laboratory diagnosis)..........................................................5
分子生物學之鑑定方法...........................................................................6
核糖體核酸內轉錄區(rRNA interal transcribed spacer, ITS).................7
研究目的...................................................................................................7
研究架構...................................................................................................7
材料與方法
實驗用菌株、培養及保存........................................................................9
DNA萃取(DNA extraction)....................................................................10
ITS區域的增幅及定序...........................................................................11
分子選殖(Molecular cloning).................................................................12
資料庫建立.............................................................................................14
探針設計.................................................................................................14
晶片製備.................................................................................................16
增幅ITS區域以進行晶片雜合反應......................................................16
晶片雜合反應.........................................................................................16
16S rDNA定序.....................................................................................18
靈敏度(sensitivity)及特異性(specificity)定義.......................................18
結果
探針篩選.................................................................................................19
Sense及antisense探針..........................................................................19
探針不配對(mismatch)核苷酸的設計………………………………...19
菌種特異性探針(Species-specific probe)及雜合反應結果之判讀......20
菌群特異性探針(Group-specific probe).....................................21
以寡核苷酸晶片鑑定參考菌株結果…………………………………22
以寡核苷酸晶片鑑定臨床菌株結果…………………………………23
DNA晶片之靈敏度及特異性................................................................26
討論
Bacteroides spp.之鑑定...........................................................................27
厭氧革蘭氏陰性桿菌之鑑定.................................................................28
Veillonella spp.之鑑定............................................................................29
 Clostridium spp.之鑑定..........................................................................30
厭氧革蘭氏陽性球菌、不產孢桿菌之鑑定...........................................30
以寡核苷酸晶片鑑定厭氧菌之展望.....................................................31
參考文獻.........................................................................................................33
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