臺灣博碩士論文加值系統

傳統鑑定分枝桿菌的方法既耗時又需要經驗豐富的人員才能正確鑑定，因此，本研究欲利用real-time PCR加上high-resolution melting curve analysis (HRM) 的方式來快速區分臨床上常見的分枝桿菌。首先，將16種臨床常見之分枝桿菌菌種（包括M. tuberculosis (MTB)、 M. asiaticum、M. gordonae、M. kansasii、M. szulgai、 M. avium、M. fortuitum、M. chelonae、M. intracellulare、 M. marinum、 M. terrae、M. xenopi、M. scrofulaceum、M. abscessus、M. haemophilum以及M. triviale）以real-time PCR搭配HRM作分析，再與M. kansasii形成heteroduplex後，以這16種臨床常見分枝桿菌之溶解曲線圖形建立起資料庫。利用blind test的方式測試臨床108株分枝桿菌菌株的DNA，並比對資料庫的正確性。從實驗結果發現，用此法完整建立16種分枝桿菌菌種資料庫，以blind test的方式鑑定臨床菌種共108株分枝桿菌菌株，有91株跟原先鑑定結果一致，正確率為84%。有9株菌株無法判斷其菌種，另有8株與原先鑑定結果不一致，但可用傳統的菌落生長速度以及照光產色的特性來輔助觀察菌種正確性。因此，此方法既快速、不需要probes，花費少，對於臨床上快速區分常見的分枝桿菌提供了一個良好的方法。

The conventional way for mycobacterial species identification usually requires an experienced personal and is time-consuming. In this study, we explored the combined use of real-time PCR and high-resolution melting (HRM) curve analysis for rapid detection and identification of clinically important mycobacterial species. At first, the HRM profiles for sixteen mycobacterial species, including M. tuberculosis (MTB), M. asiaticum, M. gordonae, M. kansasii, M. szulgai, M. avium, M. fortuitum, M. chelonae, M. intracellulare, M. marinum, M. terrae, M. xenopi, M. scrofulaceum, M. abscessus, M. haemophilum and M. triviale, were established using CAP standard mycobacterial isolates. All these 16 species can be identified by the HRM profiles after heteroduplex formation with M. Kansasii. To further extend the clinical application of this method, 108 clinical mycobacterial isolates were subjected to a blind HRM analysis. The results showed that the species of 91 isolates were correctly identified with the accuracy rate equivalent to 84%. Eight of the isolates were not identified correctly, although all of them can be identified by traditional characteristics of growth rate and pigmentation. In addition, 9 isolates can not be identified due to the failure of PCR. Together, this method is rapid and cost-effective without using probe for detection. Hence, the combined use of real-time PCR and high-resolution melting curve analysis offers a good way for rapid identification of clinical mycobacteria in a clinical setting.

指導教授推薦書…………………………………………………………………
口試委員會審定書………………………………………………………………
授權書……………………………………………………………………………. iii
誌謝……………………………………………………………………………… vi
中文摘要…………………………………………………………………………… v
英文摘要…………………………………………………………………………. vi
目錄……………………………………………………………………………... vii
第一章 緒論………………………………………………………………………... 1
1.1 分枝桿菌簡介……………………………………………………...……… 1
1.2 非結核性分枝桿菌簡介…………………………………………...……… 2
1.2.1非結核性分枝桿菌的分類…………………………………………… 2
1.3 傳統上鑑定分枝桿菌的方法…………………………………...………… 3
1.4 分子診斷法……………………………………………………………...… 4
1.5 選用16S rRNA gene當作target的原因…………………………………. 6
1.6 實驗目的……………………………………………………….….….…… 6
第二章 材料與方法………………………………………………………………... 7
2. 1資料庫菌種來源…………………………………………………….……… 7
2.1.1 CAP標準菌種………………………………………………………… 7
2.1.2 臨床菌種……………………………………………………………. 7
2.1.3 Blind test臨床菌株種類及數量…………………………………….. 7
2.2 實驗試劑及儀器…………………………………………………………. 8
2.3 Target gene的選擇……………………………………………………… 9
2.4 Real-time PCR中DNA template的取得………………………………. 10
2.4.1 CAP菌株的real-time PCR template DNA的取得…………………. 10
2.4.2 臨床菌株的real-time PCR template DNA的取得……………….. 10
2.5 Real-time PCR搭配high-resolution melting curve analysis的方法 11
2.5.1 用lightcycler real-time PCR放大16S rRNA gene……………… 11
2.5.2 high-resolution melting curve acquisition and analysis…………… 11
2.5.3 Heteroduplex formation……………………………………………… 12
2.5.4 建立資料庫…………………………………………………...….. 12
2.6 Sequencing analysis for CAP species………………………………….. 12
2.7 Blind test……………………………………………………………….. 13
第三章 實驗結果…………………………………………………………………. 14
3.1選擇16S rRNA gene上適合的target gene…………………….….……… 14
3.2分枝桿菌菌種資料庫的建立……………………………………………... 14
3.3定序以確認資料庫中各分枝桿菌菌種的正確性………………………… 16
3.4臨床分枝桿菌的blind test測試…………………………………..………. 16
第四章 結果討論…………………………………………………………………. 19
參考文獻…………………………………………………………………….……… 22
附圖 ………………………………………………………………………………. 27
圖A 92年到95年林口長庚醫院院內所檢測出結核菌群的比例………… 27
圖B Runyon分群……….…………………………………………………….. 28
圖C 傳統生化試驗………………………………………………………….. 29
圖D 92年到95年林口長庚醫院臨床常見的分枝桿菌菌種……………….. 31
Table 1 臨床常見非典型分枝桿菌的16sRNA target gene的序列分群………. 32
Table 2 Blind test………………………………………………………………... 33
Table 3 臨床分枝桿菌菌株之操作結果與原先鑑定結果……………………. 34
圖一 Target: 16S rRNA gene from 71 to 226bp………………………………… 36
圖二 16種分枝桿菌的melting plot及derivative plot…………………………… 37
圖三 CAP分枝桿菌菌種圖形之資料庫……………………………………. 38
圖四 利用另一個target-ITS來區分M. abscessus及M. chelonae………………. 39
圖五 利用HRM及形成heteroduplex鑑定未知純化菌種之流程圖…………...40
圖六 臨床分枝桿菌菌種圖形之資料庫……………………….………… 41
圖七 Blind test臨床分枝桿菌菌株無法判讀菌種之derivative plot……………42
圖八 Blind test臨床分枝桿菌菌株與結果不一致之derivative plot……………43
圖九 與reference M. kansasii 形成heteroduplex 之blind test臨床菌株……44
圖十 臨床上用HRM及形成heteroduplex快速鑑定未知臨床菌種的流程圖45
圖十一Sequencing for clinical M. gordonae (unknown isolate no. 45) …………..46
附錄 定續結果…………………………………………………………………47

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林碧芬、吳竹蘭、彭建芳、薛博仁、蔡文城 鄧麗珍、蘇維鈞，《結核菌檢驗手冊》，出版年月:中華民國九十三年三月