Study on one-tube LAMP-PCR-hybridization-thermalmelt-ELISA method for molecular detection of multidrug resistance in Mycobacterium tuberculosis isolates

• Main Authors: Ching-Yu Chen, 陳靜瑜
• Other Authors: Chien-Fang Peng
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/02717507634594507392

碩士 === 高雄醫學大學 === 醫學檢驗生物技術學研究所 === 98 === 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.