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標題: 以PCR-DGGE方法分析環境中非結核性分枝桿菌
Identification of Non-Tuberculous Mycobacterium in Environmental Samples by PCR-Denaturing Gradient gel Electrophoresis
作者: 王郁芬
Wang, Yu-Fen
關鍵字: Nontuberculous mycobacteria (NTM);非結核性分枝桿菌(NTM);Denaturing Gradient Gel Electrophoresis (DGGE);16S rRNA;heat shock protein gene (hsp gene);變性梯度明膠電泳(DGGE);16S rRNA;熱休克蛋白基因(hsp gene)
出版社: 環境工程學系
非結核性分枝桿菌(NTM)容易對於免疫功能較差的人造成一些疾病;有些菌種在人類或動物身上所引起的症狀和結核病相似,有極少數的報告指出NTM會藉由人與人之間接觸傳染,已有文獻指出,目前台灣地區之自來水中已有非結核性分枝桿菌的存在,特別是在於醫療院所之配水系統中或飲用水中,現今國內外對於環境中非結核性分枝桿菌的研究,尚未有一套公認且完整的快速鑑別方法,因此,本研究利用PCR-DGGE之分子生物分析方法,以分離鑑別目前台灣地區環境中非結核性分枝桿菌族群,並建立一套目前適合台灣地區環境中非結核性分枝桿菌族群的分析方法,進一步突破傳統分析上耗時和不精確的困難。以PCR-DGGE方法分析環境中非結核性分枝桿菌,目前國內外文獻並未提及且沒有研究使用,此分析方法針對環境中非結核性分枝桿菌族群的分析,本實驗室已將此分析方法最佳化,不但快速,菌種間有良好的分離效果。本研究以16S rRNA和hsp gene進行非結核性分枝桿菌菌種鑑別,目前在經過培養的自來水中已分離出30株菌株,經由PCR放大有20株菌株為正訊號,經DGGE分析後共分離出五種不同之單一非結核性分枝桿菌菌種,包含M. porcinum, Mycobacterium sp., M. lacticola, M. mucogenicum, M. wolinskyi。證明以PCR-DGGE方法分析,無論是利用16S rRNA或是hsp gene進行菌種鑑別,皆可有良好的分析效果,且DGGE指紋圖譜相對而言較RFLP的指紋圖譜容易建立,對於非結核性分枝桿菌族群的判別上增加了許多分析上的優勢。

Nontuberculous mycobacteria (NTM) is a common microorganisms which cause diseases in immunocompromised patients. In addition, Some species may cause infections similar to tuberculosis in humans and animals.There is very limited documentation of person-to-person transmission of NTM. This aim of this study is to develop a method which can detect the NTM of the tap water of hospital. In our study, we try to combine some molecular techniques based on the 16S rRNA and the hsp gene of mycobacteria, PCR-DGGE,for identification of NTM in environmental samples.
30 pure cultures were applied in this research, but only 20 cultures showed the postive signal after PCR analysis. Five different species of NTM were identified form these 20 cultures after 16S rRNA sequencing , including M. porcinum, Mycobacterium sp., M. lacticola, M. mucogenicum, M. wolinskyi. These results demostrated the method we developed that identification nontuberculosis mycobacterium in environmental samples by PCR-DGGE has been established and more precise. In the future, we hope this efficient method could be use for definitive detection of NTM of environmental samples.
Appears in Collections:環境工程學系所

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