結核桿菌快速診斷晶片之研發

Development of a diagnostic gene chip for Mycobacterium tuberculosis

孫嘉穗(Chia-Sui Sun)

指導教授 ： 鍾飲文

高雄醫學大學/醫學院/醫學研究所/碩士(2008年)

https://doi.org/10.6832/KMU.2008.00045

摘要

依據世界衛生組織之預估，結核桿菌約已感染了全球三分之ㄧ的人口，並且統計每年大約新增八百萬新病例且造成兩百萬人因結核病死亡，而結核病也是台灣地區罹患人數最多、致死率最高之法定傳染病，在公共衛生上是相當嚴重之議題。許多研究指出，有效控制結核病端賴早期診斷以及有效抗結核治療。然而，截至目前為止，臨床上結核病的診斷大多仍以患者的臨床表徵、組織病理學、抗酸菌染色和培養等技術為主。由於這些方法受限於檢驗時效，靈敏度及特異性，嚴重影響臨床上結核病患者的診治成效。近年來由於分子生物學的蓬勃發展，利用相關分子檢測技術終於讓結核病的臨床診斷有了革命性的進步。然而目前均僅可分型至結核桿菌群，無法更一步直接偵測出結核桿菌的存在，或是僅針對單一基因標的檢測，在臨床的診斷上，無法達到令人滿意的準確性。因此，本研究利用菌體中差異性區段（Regions-of-difference；RD）選出特異性目標基因，以此作為結核桿菌特異性檢測標的，不但保有分子診斷技術快速敏感性高的優點，亦可彌補傳統分子檢測方式無法檢測至結核桿菌之缺點。首先，我們將臨床上收集之痰液檢體以傳統分子檢測方式進行聚合酶連鎖反應、聚合酶連鎖反應結合限制酵素切割片段長度多型性作分子分型，並分析其中結核桿菌群、非結核分支桿菌及其他菌株之間分佈的比例。爾後我們利用所選定之目標基因群以聚合酶連鎖反應建立在結核桿菌標準菌株之聚合酶連鎖反應圖譜，作為判定結核桿菌之依據。接下來利用目標基因群在基因陣列尼龍膜片建構原型結核桿菌檢測平台，並以標準菌株測試，比對目標基因群在聚合酶連鎖反應圖譜與原型結核桿菌檢測平台呈色結果是否相符，修正反應條件，最後建立以痰液檢體之結核桿菌快速診斷晶片。此快速診斷平台的建立，可作為未來提供臨床醫師在臨床篩檢結核病患者之輔助工具，達到早期診斷、早期治療、有效控制結核病之目標，而這也將為結核病患者的診治開啟嶄新的一頁。

關鍵字

結核桿菌 ； 呈色型晶片 ； 分子診斷

並列摘要

Tuberculosis（TB）is an old-age, infectious disease that has spread to nearly every corner of the world. The global incidence rate of TB is increasing by approximately 0.4% per year. Therefore, it is a critical issue to alleviate the growing worldwide TB epidemic for public health. The most efficient way of controlling TB lies in early diagnosis and effective TB chemotherapy. At present, clinical symptoms, histopathological features, acid-fast stain, bacillary morphology are mainly the ways of diagnosing TB in developing countries. Recent biotechnological advance has fuelled a revolution in the diagnosis of tuberculosis but is still limited that each genetic marker must be detected separately. Moreover, using PCR and PCR－RFLP can only detect the presence of Mycobacterium tuberculosis complex but not Mycobacterium tuberculosis. Thus, it is important to develop an easier and more effective method that can directly detect the presence of Mycobacterium tuberculosis in sputum sample which may be of great value in TB control. First, 246 sputum samples were obtained from Kaohsiung Medical University Hospital and applied PCR and PCR－RFLP to genotype each of the Mycobacterium species. Furthermore, we analyzed the distribution of Mycobacterium tuberculosis complex, Nontuberculous Mycobacterium and other species in Taiwan. In order to directly identify the existence of Mycobacterium tuberculosis, we selected 14 candidate genes within the Regions-of-Difference （RD） of the bacteria and used PCR to construct the Mycobacterium tuberculosis pattern as an reference to detect Mycobacterium tuberculosis in Mycobacterium tuberculosis complex. Afterwards, we constructed the prototype of the diagnostic chip with the same candidate genes and compared the pattern of membrane array with the PCR pattern. After conditional adjustments, the pattern of the diagnostic chip was fully matched with the PCR pattern of the candidate genes. Finally, we successfully constructed the diagnostic gene chip for direct detection for Mycobacterium tuberculosis. With the establishment of the diagnostic gene chip, it can speed up the whole process of diagnosing tuberculosis patients and may have a great potential for clinical applications.

並列關鍵字

Mycobacterium tuberculosis ； membrane array ； molecular diagnosis

參考文獻

1. 世界衛生組織(WHO) http://www.who.int/tb/en/.

2. 衛生署疾病管制局 http://203.65.72.83/.

3. Zhou YM, Yang RQ, Tao SC, Li Z, Zhang Q, Gao HF, Zhang ZW, Du JY, Zhu PX, Ren LL, Zhang L, Wang D, Guo L, Wang YB, Guo Y, Zhang Y, Zhao CZ, Wang C, Jiang D, Liu YH, Yang HW, Rong L, Zhao YJ, An S, Li Z, Fan XD, Wang JW, Cheng Y, Liu O, Zheng Z, Zuo HC, Shan QZ, Ruan L, Lu ZX, Hung T, Cheng J: The design and application of DNA chips for early detection of SARS-CoV from clinical samples. J Clin Virol 2005;33:123-131.

4. Kanduma E, McHugh TD, Gillespie SH: Molecular methods for Mycobacterium tuberculosis strain typing: a users guide. J Appl Microbiol 2003;94:781-791.

5. Juan Carlos Palomino SCL, Viviana Ritacco: Tuberculosis 2007 2007:p1-687.