- 學位論文
利用單支試管呈色法LAMP-PCR-hybridization-thermal melt-ELISA method偵測結核菌的多重抗藥性之研究
Study on one-tube LAMP-PCR-hybridization-thermal melt-ELISA method for molecular detection of multidrug resistance in Mycobacterium tuberculosis isolates
摘要
結核病(Tuberculosis, TB)是全世界人口死亡的原因之一。新增加的TB病例在東南亞地區每年仍然持續的出現。近年來，多重抗藥性 (multidrug-resistant, MDR) 和超級抗藥性 (extensively drug-resistant, XDR) 結核病已經成為全球的重大問題。 本研究的目的是建立一種快速、簡便、準確的分子診斷技術應用於多重抗藥性結核菌的偵測。利用單支試管呈色法One-tube LAMP-PCR-thermal melt- hybridization -ELISA偵測多重抗藥結核菌是本研究的目標。其原理是使用微量平盤核酸序列基礎的LAMP (loop-mediated isothermal amplification, LAMP-PCR）聚合酶連鎖反應酵素免疫法針對多重抗藥結核菌抗藥性相關之各種基因之突變與正常DNA序列之差異性，設計出各組之引子利用LAMP-PCR技術得到PCR產物，並搭配熱熔解分析（thermal melt assay），利用同質性雙股雜交鍵結(homoduplex) (高熔解度)和異質性雙股雜交鍵結(heteroduplex) (低熔解度) 的熔解溫度差異，將和wild-type特異性探針形成的異質性雙股雜交鍵結(有變異點)的雜交鍵結經加熱熔解後被洗去，剩下熔解溫度較高的同質性雙股雜交鍵結的結合，後續接著進行ELISA（enzyme-linked immunosorbent hybridization ，酵素結合免疫吸附法）呈色反應完成偵測。有呈色反應的為不具抗藥結核菌的基因序列，反之，無呈色反應則表示為抗藥結核菌，具有變異點。 抗藥性相關之各種基因包括Isoniazid 抗藥性是 katG, inhA, 和mabA promoter。Rifampin抗藥性是rpoB。 Amikacin抗藥性是rrs 。Fluoroquinolones抗藥性是gyrA和 gyrB。本研究利用One-tube LAMP-PCR-hybridization-thermal melt-ELISA分析對isoniazid, rifampin, amikacin及fluoroquinolones具抗藥性之結核菌與傳統藥物敏感性試驗比較敏感度分別為92.2%、94.7%、91.3％和92.9%，而特異性都可以達到100%，最後檢測效能分別為95.2%、98.6%、98.6％和99.3%，足見本研究所採用之One-tube LAMP-PCR- hybridization -thermal melt-ELISA對變異熱點的檢測精準度極高，值得應用於臨床上的檢驗。
並列摘要
In this study, we design a simple and rapid colorimetric detection method, One- tube LAMP-PCR-thermal melt- hybridization –ELISA, to detect resistance to isoniazid, rifampin, amikacin and fluroquinolone in M. tuberculosis isolated from clinical specimens. The clinical performance of this method for detecting resistance of isoniazid, rifampin, amikacin and fluroquinolone in M. tuberculosis isolates showed 95.2％, 98.6%, 98.6% and 99.3%, respectively. This method had sensitivity of 92.2%, 94.7%, 91.3% and 92.9% for detecting resistance of isoniazid, rifampin, amikacin and fluroquinolone in the culture method. This method for detection of resistance in M. tuberculosis isolates is a convenient method to be performed within a one working day. One- tube LAMP-PCR-thermal melt- hybridization –ELISA method was designed based on hot spot point mutation in target drug-resistant gene, using LAMP-PCR, using thermal melt for differentiating homoduples (wild type) and heteroduples (mutant), then hybridization, and colorimetric method with ELISA. In this study, LAMP assay is used to amplify DNA from drug-resistant M. tuberculosis and ELISA is used for the colorimetrical determination. This method will be a useful tool for rapid diagnosis of hot spot point mutation in isoniazid resistance genes of katG, inhA and mabA promoter, rifampin resistance gene of rpoB, fluoroquinolones resistance genes of gyrA and gyrB, amikacin resistance gene of rrs in M. tuberculosis isolates.
參考文獻
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