A rapid fluorescence polarization-based method for genotypic detection of drug resistance in Mycobacterium tuberculosis

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• 作者：Yisuo Sun (1) (2)
  Shufen Li (3)
  Lin Zhou (4)
  Lin Zhou (3)
  Qiu Zhong (4)
  Shisong Fang (5)
  Tao Chen (4)
  Lijun Bi (6)
  Wai-Kin Mat (1)
  Cunyou Zhao (1) (3)
  Hong Xue (1) (2)
  
• 关键词：Drug resistance ; Genotyping ; M. tuberculosis ; One Label Extension ; DNA diagnostics
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2014
• 出版时间：May 2014
• 年：2014
• 卷：98
• 期：9
• 页码：4095-4105
• 全文大小：394 KB
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• 作者单位：Yisuo Sun (1) (2)
  Shufen Li (3)
  Lin Zhou (4)
  Lin Zhou (3)
  Qiu Zhong (4)
  Shisong Fang (5)
  Tao Chen (4)
  Lijun Bi (6)
  Wai-Kin Mat (1)
  Cunyou Zhao (1) (3)
  Hong Xue (1) (2)
  
  1. Division of Life Science and Applied Genomics Center, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
  2. Nano Science and Nano Technology Program, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
  3. Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
  4. Antituberculosis Research Institute of Guangdong Province, Guangzhou, 510630, China
  5. Division of Microbiology Test, Shenzhen NanShan Centre for Disease Control and Prevention, Shenzhen, 518055, China
  6. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
  
• ISSN：1432-0614

文摘

Rapid detection of drug-resistant Mycobacterium tuberculosis is critical to the effective early treatment and prevention of the transmission of tuberculosis. However, conventional drug susceptibility tests for M. tuberculosis require up to several weeks. In the present study, the One Label Extension genotyping method was adapted for rapid detection of drug resistance-associated sequence variations in six genes of M. tuberculosis, viz. rpoB, rpsL, rrs, embB, katG, or inhA. The method utilizes polymerase chain reaction amplified fragments of the drug resistant genes as reaction templates, and proceeds with template-directed primer extension incorporating a fluorescence-labeled nucleotide, which is then measured by fluorescence polarization. A total of 121 M. tuberculosis isolates from clinical sputum specimens were examined by this genotyping method and verified by direct sequencing of polymerase chain reaction amplicons harboring previously reported mutational sites associated with M. tuberculosis drug resistance. Based on phenotyping results obtained from microbiology-based drug susceptibility tests, the sensitivity, specificity, and test efficiency estimated for One Label Extension assays were respectively 83.9?%, 95.5?%, and 92.4?% with ropB in rifampin resistance, 67.3?%, 97.1?%, and 84.3?% with rpsL and rrs in streptomycin resistance, 60.0?%, 96.0?%, and 91.4?% with embB in ethambutol resistance, 68.4?%, 94.9?%, and 86.3?% with inhA and katG in isoniazid resistance, and 74.1?%, 98.9?%, and 93.2?% in multiple drug resistance defined as resistance to at least both isoniazid and rifampin. In conclusion, examination of clinical sputum specimens by One Label Extension based genotyping provides a valid method for the rapid molecular detection of drug-resistant M. tuberculosis.