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系統識別號 U0026-0402201016032000
論文名稱(中文) 以DNA晶片鑑定結核桿菌及非結核分枝桿菌
論文名稱(英文) Identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria by an oligonucleotide array
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 98
學期 1
出版年 99
研究生(中文) 鄭伊珊
研究生(英文) Yi-Shan Jenh
學號 t3696102
學位類別 碩士
語文別 中文
論文頁數 101頁
口試委員 指導教授-張長泉
口試委員-吳俊忠
口試委員-周如文
口試委員-盧柏樑
口試委員-柯文謙
中文關鍵字 結核病  分枝桿菌  鑑定 
英文關鍵字 tuberculosis  mycobacteria  identification 
學科別分類
中文摘要 結核病(tuberculosis)在全球每年約有920萬新案例,造成約170萬人死亡,是全世界公共健康的大問題。最近三年(95-97)台灣每年有14500-16500肺結核病例(63-73例/10萬人口)。傳統方法分離及鑑定分枝桿菌,約需2至8星期。正確及快速地鑑定分枝桿菌,對病人的治療及管理很重要。本研究之目的在開發寡核苷酸晶片(oligonucleotide array)以鑑定分枝桿菌,包括6種屬於Mycobacterium tuberculosis complex (MTBC)的細菌和19種較常見的非結核分枝桿菌(nontuberculous mycobacteria, NTM)。由細菌的16S-23S rRNA基因間的intergenic spacer ( ITS )區域及gyrB基因設計探針,把探針固定在尼龍膜上,與標識毛地黃素(digoxigenin) 的PCR產物雜合反應(hybridization)後,再以標記鹼性磷酸酶(alkaline phosphatase)的抗體呈現反應結果。經測試250株(88株參考菌株及162株臨床菌株)目標菌株(target strain)及73株非目標菌株(nontarget strain)後,晶片鑑定的靈敏度(sensitivity)和特異性(specificity)分別為98.8% (246/249)及98.6% (72/73),且MTBC中所有菌種均可被正確鑑定(M. africanum及M. pinnipedii除外)。晶片的檢測極限(detection limit)為每測試(assay)中含50 fg (M. tuberculosis H37Rv)或500 fg (M. abscessus CCUG 20993T)的DNA。
以晶片檢測205個BACTEC MGIT 960 (Becton Dickinson Biosciences Microbiology Products, Sparks, MD, USA)陽性培養管中的分枝桿菌,再和經次培養(subculture)並以PCR restriction endonuclease assay (PRA)鑑定的結果比較,在75個以RFLP檢測到MTBC的培養管中,晶片均檢測到MTBC。在12個以RFLP檢測不到MTBC的培養管中,晶片可以檢測到M. tuberculosis。有37個MGIT管以晶片檢測到含二種以上的分枝桿菌(RFLP均只有檢測到一種細菌) ,另有43管晶片鑑定的結果和RFLP的方法不符合,這些含二種以上的分枝桿菌和不符合的樣品正以第三種方法分析中。
以晶片直接檢測20個經消化去汙染檢體(12個痰、4個肺泡沖洗液、及3個其他檢體)中的分枝桿菌,其中14個抗酸性染色陽性,6個陰性。並和AmplicorTM PCR Mycobacterium tuberculosis test (Roche Diagnostic Systems, Inc., Branchburg, NJ, USA)的結果做比較。在這20個檢體中AmplicorTM 系統檢測到18個含有MTBC,而晶片偵測到19個含有M. tuberculosis (或MTBC),且其中16個檢體含有二種或以上分枝桿菌。由於晶片對M. tuberculosis的檢測極限(50 fg/assay,等於103 CFU/ml),靈敏度約等於抗酸性染色的10倍(104 CFU/ml),除了純菌以外,預期晶片可以直接檢測臨床檢體中的分枝桿菌,但仍需測更多的檢體以確認。晶片的雜合檢測時間約為8小時。
英文摘要 Infections caused by mycobacteria have been a serious health problem worldwide. Mycobacterium tuberculosis complex (MTBC) is the major causative agent of human tuberculosis that causes about 9.2 million new cases and 1.7 million deaths every year in the world. Antimicrobial agents used to treat infections caused by MTBC or nontuberculous mycobacteria (NTM) are quite different. In addition, different species of NTM may have different antimicrobial susceptibility. Therefore, rapid identification of Mycobacterium to the species level is of clinical importance. Conventional methods for the isolation and identification of Mycobacterium species are time-consuming and the results can be inconclusive. The aim of this study was to develop an oligonucleotide array that could differentiate members in MTBC and could identify 19 species of NTM. Specific oligonucleotide probes (17 to 27 nucleotides) were designed from the 16S-23S rRNA gene intergenic spacer (ITS) region and the gyrB gene. The identification method consisting of PCR amplification of the ITS region and gyrB gene, followed by hybridization of the digoxigenin-labeled PCR product to probes immobilized on nylon membrane. A collection of 250 target strains (88 reference strains and 162 clinical isolates) and 73 nontarget strains and the sensitivity and specificity of the array were found to be 99.2% (247/249) and 98.6% (72/73), respectively. The detection limit of the array for M. tuberculosis H37Rv was about 50 fg genomic DNA per assay (equivalent to 12 cells) and was 500 fg genomic DNA per assay for M. abscessus CCUG 20993T.
The array was further used to detect mycobacteria in 205 BACTEC MGIT-positive tubes (Mycobacteria Growth Indicator Tube, Becton Dickinson Biosciences Microbiology Products, Sparks, MD, USA) and the results were compared with those obtained by subculture on 7H11 agar followed by identification made by the technique of PCR restriction fragment length polymorphism (PRA). For 75 tubes that were found to contain MTBC by PRA, all of them were also positive for M. tuberculosis or MTBC by the array. However, the array detected an addition 12 tubes containing MTBC. In addition, 37 tubes were found to contain two or more Mycobacterium species by array hybridization, but the subculture/PRA process usually identified a single species. In 43 tubes, the species identified by PRA was different from that detected by the array. A third method will be used to verify the discrepant identifications between PRA and the array and to verify the presence of multiple species in some MGIT tubes.
Finally, the array also was used to directly detect mycobacteria in 20 clinical specimens including 12 sputum samples, 5 bronchoalveolar lavage samples and 3 other specimens; among these specimens, 14 were acid-fast stain positive and 6 were negative. The results obtained by the array were compared with those obtained by AmplicorTM PCR Mycobacterium tuberculosis test (Roche Diagnostic Systems, Inc., Branchburg, NJ, USA). The AmplicorTM system detected MTBC in 18 specimens, but the array detected M. tuberculosis or MTBC in 19 samples. It was interesting to find that among the 19 array-positive samples, 16 contained more than one Mycobacterium species. Our study clearly indicated that a high percentage of patients infected with MTBC is also co-infected with other mycobacteria. In conclusion, the array can be used to identify pure mycobacterial cultures, mycobacteria in MGIT (+) tubes, and have a great potential to directly detect Mycobacterium in clinical specimens. The whole procedures of array hybridization took about 8 h.
論文目次 中文摘要.......................................................................................................Ⅰ英文摘要..................................................................................................Ⅲ
誌謝.............................................................................................................. Ⅴ
目錄...............................................................................................Ⅵ
表目錄...........................................................................................................Ⅸ
圖目錄.......................................................................................................ⅩⅠ
緒論..................................................................................................................1
分枝桿菌屬(Mycobacterium species) ……………………………………1
結核菌群(Mycobacterium tuberculosis complex, MTBC) ……………...2
非結核分枝桿菌(Nontuberculous mycobacteria, NTM)…………… …..3
實驗室診斷(Laboratory diagnosis)及分子生物學鑑定方法 …………...3
分枝桿菌流行病學 ………………………………………………………5
研究目的 ………………………………………………………………....6
研究架構 …………………………………………………………………7
材料與方法.......................................................................................................8
實驗用菌株、菌株的培養保存及菌種DNA ……………………….......8
臨床檢體MGIT (Mycobacterial Growth Indicator Tubes)及痰抹片陽性之檢體 (smear positive sputum) ………………………………………....9
DNA萃取 (DNA extraction) …………………………………………..10
熱休克蛋白65聚合酶連鎖反應及限制酶分析(hsp65 PCR-restriction analysis, PRA)……………………………………………………...11
ITS區域 (ITS region)及gyrB基因之增幅及定序 ……………………11
特異性探針(specific probes)之設計 …………………………………...12
分子選殖(Molecular cloning) ………………………………………….14
以製造點突變(Site direct mutation)方式產生模擬產物 ………………16
分枝桿菌晶片製備 ……………………………………………………..16
分枝桿菌晶片的雜合反應 (chip hybridization) ………………………17
晶片雜合反應之結果判讀 ……………………………………………..18
ITS, 16S rRNA基因及gyrB之定序 ………………………………….19
靈敏度、特異性、偵測極限、陽性預測值以及陰性預測值之定義
………………………………………………………………………….20
結果 .................................................................................................................21
分枝桿菌特異性探針 ………………………………………………….21
正股探針(sense probe)及負股探針(anti-sense probe) …………………21
Mycobacterium spp.正對照組(positive control, PC)探針 ……………...21
結核菌群(MTBC)之鑑定 ………………………………………………22
非結核分枝桿菌(NTM)之鑑定 ………………………………………...24
參考菌株之晶片測試 …………………………………………………..24
臨床菌株之晶片測試 …………………………………………………..26
臨床中分枝桿菌之直接鑑定 …………………………………………..27
分枝桿菌晶片之靈敏度(sensitivity)、特異性(specificity)、陽性預測值(PPV)及陰性預測值(NPV) …………………………………………….28
晶片之偵測極限(detection limit) ………………………………………29
在其他NTM存在下檢測M. tuberculosis……………………………29
討論 ..................................................................................................................30
雙套式聚合酶連鎖反應(duplex PCR) …………………………………30
晶片鑑定結果不一致之菌種 …………………………………………..30
以晶片直接檢測臨床檢體之分枝桿菌 ………………………………..31
參考文獻…………………………………………………………..………...33


表目錄
TABLE 1. Comparison of commercial kits for identification of mycobacteria…………………………………………………….39
TABLE 2. Target species of Mycobacterium tuberculosis complex used in this study……………………………………………………………….........42
TABLE 3. Target nontuberculous mycobacteria used in this study.…...........45
TABLE 4. Nontarget strains used for specificity test of the array.……..........49
TABLE 5. Results of identification of M. tuberculosis complex by array hybridization.………………………...…………………………..53
TABLE 6. Mycobacterium species and their species-specific identification probes on the array.……………………………………………...56
TABLE 7. Results of identification of nontuberculous mycobacteria by array hybridization.………………………………………..…………..58
TABLE 8. Analysis of discrepant identifications produced by the array.…………………..………………………………………...64
TABLE 9. Detection of mycobacteria in BACTEC Mycobacteria Growth Indicator Tube (MGIT 960)-positive cultures by the array.……………………………………………………………66
TABLE 10. Concordant identifications of Mycobacterium from BACTEC Mycobacteria Growth Indicator Tube (MGIT 960)-positive cultures as determined by the array and by subculturing on 7H11 agar followed by identification with the technique of hsp65 PCR-restriction assay (PRA).……………………………...........69
TABLE 11. Disconcordant identifications of Mycobacterium from BACTEC Mycobacteria Growth Indicator Tube (MGIT 960)-positive cultures as determined by the array and by subculturing on 7H11 agar followed by identification with the technique of hsp65 PCR-restriction assay (PRA).…………………………………...78
TABLE 12. Detection of Mycobacterium from acid fast stain-negative specimens and from BACTEC Mycobacteria Growth Indicator Tube (MGIT 960)-negative cultures as determined by the array and by subculturing on 7H11 agar followed by identification with the technique of hsp65 PCR-restriction assay (PRA).……...…...81
TABLE 13. Direct detection of mycobacteria in clinical specimens by the array……………………………………………………………..82



圖目錄

FIG. 1. Alignment of gyrB gene of Mycobacterium bovis and other species in the M. tuberculosis complex.………….........................................85
FIG. 2. Alignment of gyrB gene of Mycobacterium canettii, M. tuberculosis, and other species in the M. tuberculosis complex.………....86
FIG. 3. Layout of oligonucleotide probes on the array.……………………...87
FIG. 4. The step by step procedures of array hybridization..……..........….....88
FIG. 5. Hybridization results of Mycobacterium tuberculosis complex..........89
FIG. 6. Hybridization results of nontuberculous mycobacteria (NTM).…….90
FIG. 7. The procedures of mycobacterial identification at KMUH..…….......92
FIG. 8. A selection of chips showing the discrepant identifications of Mycobacterium from MGIT (+) cultures as determined by the array and by subculturing on 7H11 agar followed by identification with PRA………………………………………………….93
FIG. 9. Direct detection of mycobacteria in 20 clinical specimens..…….......97
FIG. 10. The detection limit of probes MTBC1 and MTBC3 used to identify Mycobacterium tuberculosis H37Rv was 10 fg genomic DNA per microliter..……………......................................……………….99
FIG. 11. The detection limit of probes Mabs4-4 and Mfor5-1L used to identify Mycobacterium abscessus CCUG 20993T and M. fortuitum JCM 6387, respectively, was 100 fg genomic DNA per microliter....100

FIG. 12. Detection of M. tuberculosis by the chip under different ratios of cell number of M. abscessus CCUG 20997T and M. tuberculosis H37Rv………………………………………………………….101
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