title: Multi-Fluorescence Real-Time PCR Assay for Detection of RIF and INH Resistance of M. tuberculosis.
abstract: BACKGROUND
  Failure to early detect multidrug-resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes, and highlights the need to rapidly detect resistance to rifampicin (RIF) and isoniazid (INH).
  
  
  METHODS
  In Multi-Fluorescence quantitative Real-Time PCR (MF-qRT-PCR) assay, 10 probes labeled with four kinds of fluorophores were designed to detect the mutations in regions of rpoB, katG, mabA-inhA, oxyR-ahpC, and rrs. The efficiency of MF-qRT-PCR assay was tested using 261 bacterial isolates and 33 clinical sputum specimens. Among these samples, 227 Mycobacterium tuberculosis isolates were analyzed using drug susceptibility testing (DST), DNA sequencing and MF-qRT-PCR assay.
  
  
  RESULTS
  Compared with DST, MF-qRT-PCR sensitivity and specificity for RIF-resistance were 94.6 and 100%, respectively. And the detection sensitivity and specificity for INH-resistance were 85.9 and 95.3%, respectively. Compared with DNA sequencing, the sensitivity and specificity of our assay were 97.2 and 100% for RIF-resistance and 97.9 and 96.4% for INH-resistance. Compared with Phenotypic strain identification, MF-qRT-PCR can distinguish 227 M. tuberculosis complexes (MTC) from 34 Non-tuberculous mycobacteria (NTM) isolates with 100% accuracy rate.
  
  
  CONCLUSIONS
  MF-qRT-PCR assay was an efficient, accurate, reliable, and easy-operated method for detection of RIF and INH-resistance, and distinction of MTC and NTM of clinical isolates.
authors.affiliation: State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University Shanghai, China.
authors.forename: Jingfu
authors.initials: J
authors.lastname: Peng
authors.affiliation: Department of Biotechnology, School of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University Wuhan, China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Yu
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Medical School, Shanghai Pulmonary Hospital, Tongji University Shanghai, China.
authors.forename: Zhenling
authors.initials: Z
authors.lastname: Cui
authors.affiliation: State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University Shanghai, China.
authors.forename: Wenfei
authors.initials: W
authors.lastname: Xue
authors.affiliation: The Third People's Hospital of Shenzhen Shenzhen, China.
authors.forename: Ziyi
authors.initials: Z
authors.lastname: Luo
authors.affiliation: Department of Biotechnology, School of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University Wuhan, China.
authors.forename: Zilu
authors.initials: Z
authors.lastname: Wen
authors.affiliation: State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University Shanghai, China.
authors.forename: Minghua
authors.initials: M
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University Shanghai, China.
authors.forename: Danqing
authors.initials: D
authors.lastname: Jiang
authors.affiliation: Haoding Technology Limited Company Shenzhen, China.
authors.forename: Heping
authors.initials: H
authors.lastname: Zheng
authors.affiliation: State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University Shanghai, China.
authors.forename: Hai
authors.initials: H
authors.lastname: Wu
authors.affiliation: Department of Immunology and Medical Microbiology, School of Medicine, Shanghai Jiao Tong University Shanghai, China.
authors.forename: Shulin
authors.initials: S
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University Shanghai, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Li
keywords: Mycobacterium tuberculosis; drug resistance; isoniazid; quantitative real-time PCR; rifampicin