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標題: 飲用水中非結核性分枝桿菌之分子生物技術快速鑑定
作者: 洪俊雄
關鍵字: 非結核性分枝桿菌;NTM;聚合酶連鎖反應;變性梯度凝膠電泳;熱休克蛋白;PCR;DGGE;Hsp gene;Nontuberculous mycobacteria;Polymerase chain reaction;Denaturing gradient gel electrophoresis;Heat shock protein gene
出版社: 國立中興大學工學院;Airiti Press Inc.
Project: 興大工程學刊, Volume 18, Issue 3, Page(s) 199-209.
Drinking water pipe lines in developed countries had been popularly installed for many years. However, environmental microorganisms such as non-tuberculous mycobacteria (NTM) could intrude and grow inside of the pipe lines through the aging pipe junctions. NTM is a group of microorganisms which are not only commonly being found in the soil and natural water body but also are often being found in the water supply system of some medical institutes. Human diseases will likely being induced if the person drinks or contacts the NTM-contaminated water directly. This infection is particularly serious when happened on the low-resistance person or those who suffered from immunodeficiency. Currently, many developed countries have been scheduling adding NTM measurement into their biological drinking water standard. Since only total cell count and E. coli measurement are listed in the drinking water standard, there is truly a need of establishing background information of NTM existing in the drink water system of Taiwawn.
On the identification of NTM. pure culture incubation method is well accepted. However, this traditional method is not only time-consuming but also lacking accuracy. In this study, a fast NTM identification method was developed based on the newly developed molecular method. Gene targets including 16S rRNA (containing evolutionary information) and a 65 KDa heat shock protein gene (hsp) (containing uique antibiotic information) were selected for gene amplification and analysis. Molecular methods including Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis were combined for distinguishing the isolated NTM pure cultures. Optimized experiment procedure was established and it was concluded that either 16S rRNA or hsp gene could be used for this fast identification purpose. But for a more accurate identification, using hsp gene as target is more appropriate.

已開發國家的自來水管線雖然普及,但常因年久失修、管線老舊等因素使得周遭環境中的微生物例如非結核性分枝桿菌(non-tuberculous mycobacteria, NTM)入侵並在管線中滋長。NTM細菌廣泛地存在於土壤及自來水體中,亦常在醫療照護機構供水系統中被發現,飲用或接觸到受NTM污染的自來水將可能引起疾病,尤其對於抵抗力較差或是免疫不全的病患更容易造成重大傷害。許多先進國家已經陸續將非結核性分枝桿菌列入生物性水質指標項目中,但在台灣的水質品管項目中,有關細菌的監測項目仍僅有總菌數和大腸桿菌菌數,實有必要先行針對台灣地區飲用水中的NTM細菌進行背景調查,以作為未來訂定相關法規之依據。
針對NTM的鑑定,目前仍以傳統培養方法最為普遍,但檢驗過程耗時繁瑣且正確性不足,而且尚未有公認的快速鑑別方法。因此,本研究計畫嘗試以分子生物技術方式建立對NTM菌種快速鑑別方法,分別以16S rRNA(具有高度演化保守性)及65 KDa熱休克蛋白(heat shock protein, hsp)基因(免疫反應區域獨特性)作為目標序列,藉由聚合?連鎖反應(Polymerase Chain Reaction, PCR)結合變性梯度凝膠電泳(Denaturing Gradient Gel Electrophoresis, DGGE)進行分析及比對。在建立出最佳實驗條件以後,結果顯示,無論使用16S rRNA或hsp gene作為目標序列,配合PCR-DGGE皆可快速鑑別水體中是否有不同的NTM菌種存在。此外,根據本研究結果發現,使用hsp gene作為目標序列,可以更有效地鑑別出飲用水中之不同NTM菌種。
ISSN: 1017-4397
Appears in Collections:第18卷 第3期

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