結核分枝桿菌檢測晶片之臨床應用

Clinical application for Mycobacterium tuberculosis diagnostic chip

鄭家和(Chia-Ho Cheng)

指導教授 ： 卓夙航

共同指導教授 ： 林?茹(Shiu-Ru Lin)

高雄醫學大學/醫學院/醫學遺傳學研究所/碩士(2009年)

摘要

結核病是一相當古老的疾病，根據世界衛生組織（WHO）的統計每年大約新增八百萬結核病新病例且造成兩百萬人因結核病而死亡。在台灣結核病也同樣是公共衛生上一個嚴重之議題，據統計結核病在法定傳染病中佔 70 %，是全國罹患人數最多，死亡比例也最高的法定傳染病。在臨床、實驗室檢驗診斷方面主要以組織病理學、抗酸菌染色和培養等技術為主，但在檢驗時效性、敏感度、特異性皆有其限制，嚴重影響臨床上結核分枝桿菌患者診治成效。由於分子生物檢測技術的蓬勃發展，讓結核病患的臨床診斷擁有重大進步。然而臨床上目前僅能分型至結核分枝桿菌群，無法更進ㄧ步直接偵測並判斷結核分枝桿菌的存在。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建構結核分枝桿菌檢測平台，進而利用建構完成的結核分枝桿菌診斷晶片，進行大量臨床檢體篩檢，不但保有分子診斷技術快速且準確率高的優點，更能彌補傳統檢測方式無法偵測至結核分枝桿菌之缺點。首先，我們將臨床上第一階段已知的246位肺結核病患和第二階段疑似肺結核的795位病患之痰液檢體同步進行培養法、螢光抗酸性染色、聚合酶連鎖反應圖譜及聚合酶連鎖反應結合限制酶酵素切割片段長度多型性作菌株分型及結核分枝桿菌檢測晶片測試，分析比對培養法、抗酸性螢光染色與結核分枝桿菌檢測晶片配對的數據進行資料統計，研究結果為結核分枝桿菌檢測晶片之敏感度( sensitivity )為 84.94% 、特異性( specificity )為 91.45% 、準確率( accuracy )為 89.56% 、 陽性檢出率( positive predictive value, PPV )為82.70% 、陰性檢出率(negative predictive value )為 92.66%。故結核分枝桿菌檢測晶片的研發，對於結核分枝桿菌之演化，顯示了本研究論文充分的探討結核分枝桿菌群的菌株差異性及對遺傳性的基因標的做進一步的分析。更願造就更大的福祉於世界，發揮科學進步、研發脈動將能引領未來生活的昇華之可能性。

關鍵字

肺結核 ； 結核分枝桿菌 ； 呈色型尼龍膜矩陣 ； 抗酸性螢光染色

並列摘要

Tuberculosis (TB) is a old-age infectious disease. According to World Health Organization (WHO), there are 8 millions new cases and 2 millions dead by Tuberculosis. Tuberculosis is also a serious issue on publish health in Taiwan. TB is up to 70% in notifiable infectious disease. It’s the maximum number of people suffering and dying from. Currently, histopathological features, acid-fast stain and bacillary morphology are the mainly ways of diagnosing TB in clinical. But these diagnosis methods all have defects on sensitivity, specificity and time consuming, affecting the efficiency on clinical TB diagnosis. Recent biotechnological advance has fuelled a revolution in the diagnosis of tuberculosis but is still limited that each genetic marker must be detected separately. And only detect the presence of Mycobacterium tuberculosis complex but not Mycobacterium tuberculosis. Thus, in this research we were developed a platform for Mycobacterium tuberculosis diagnosis by the 14 specific genetic markers of the Regions-of-Difference (RD) of the bacteria. Use the diagnostic chip by large amount of clinical TB specimens. The advantages about quick and precise can compensate traditional diagnosis method for the defect of which can not detect the Tuberculosis complex.
First, we included the 1st stage of 246 known TB patients and 2nd stage of 795 suspected TB patients. Simultaneously, use the sputum as specimen to culture, acid-fast stain, PCR, PCR-RFLP and Mycobacterium tuberculosis diagnostic chip for test. Compare data of culture, acid-fast stain and Mycobacterium tuberculosis diagnostic chip. The result of Mycobacterium tuberculosis diagnostic chip indicated that the sensitivity, specificity, accuracy, positive predictive value, negative predictive value are 84.94%, 91.45%, 89.56%, 82.70%, 92.66%. After all, we developed the chip in order to approach the evolution of Mycobacterium tuberculosis and talk over the difference of hereditary genetic markers and bacteria. Wish the powerful diagnostic chip for TB can early apply to clinical diagnosis and speed up the whole process of diagnosing tuberculosis patients.

並列關鍵字

Tuberculosis ； Mycobacterium tuberculosis complex ； Colorimetric membrane array ； Fluorochrome acid fast stain

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/.

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