title: A Color-Reaction-Based Biochip Detection Assay for RIF and INH Resistance of Clinical Mycobacterial Specimens.
abstract: The widespread occurrence of drug-resistant Mycobacterium tuberculosis places importance on the detection of TB (tuberculosis) drug susceptibility. Conventional drug susceptibility testing (DST) is a lengthy process. We developed a rapid enzymatic color-reaction-based biochip assay. The process included asymmetric multiplex PCR/templex PCR, biochip hybridization, and an enzymatic color reaction, with specific software for data operating. Templex PCR (tem- PCR) was applied to avoid interference between different primers in conventional multiplex- PCR. We applied this assay to 276 clinical specimens (including 27 sputum, 4 alveolar lavage fluid, 2 pleural effusion, and 243 culture isolate specimens; 40 of the 276 were non-tuberculosis mycobacteria specimens and 236 were M. tuberculosis specimens). The testing process took 4.5 h. A sensitivity of 50 copies per PCR was achieved, while the sensitivity was 500 copies per PCR when tem-PCR was used. Allele sequences could be detected in mixed samples at a proportion of 10%. Detection results showed a concordance rate of 97.46% (230/236) in rifampicin resistance detection (sensitivity 95.40%, specificity 98.66%) and 96.19% (227/236) in isoniazid (sensitivity 93.59%, specificity 97.47%) detection with those of DST assay. Concordance rates of testing results for sputum, alveolar lavage fluid, and pleural effusion specimens were 100%. The assay provides a potential choice for TB diagnosis and treatment.
authors.affiliation: State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University, Shanghai 200433, P.R. China.
authors.forename: Wenfei
authors.initials: W
authors.lastname: Xue
authors.affiliation: State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University, Shanghai 200433, P.R. China.
authors.forename: Jingfu
authors.initials: J
authors.lastname: Peng
authors.affiliation: School of Biology and Pharmaceutical Engineering,Wuhan Polytechnic University, Wuhan 430023, P.R. China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Yu
authors.affiliation: Department of Medical Microbiology and Parasitology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China.
authors.forename: Shulin
authors.initials: S
authors.lastname: Zhang
authors.affiliation: The Third People's Hospital of Shenzhen, Shenzhen 518000, P.R. China.
authors.forename: Boping
authors.initials: B
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University, Shanghai 200433, P.R. China.
authors.forename: Danqing
authors.initials: D
authors.lastname: Jiang
authors.affiliation: The Third People's Hospital of Shenzhen, Shenzhen 518000, P.R. China.
authors.forename: Jianbo
authors.initials: J
authors.lastname: Chen
authors.affiliation: School of Biology and Pharmaceutical Engineering,Wuhan Polytechnic University, Wuhan 430023, P.R. China.
authors.forename: Bingbing
authors.initials: B
authors.lastname: Ding
authors.affiliation: Baio Technology Limited Company, Shanghai 200233, P.R. China.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University, Shanghai 200433, P.R. China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Li
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D024901:Drug Resistance, Multiple, Bacterial; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology*; D046228:Microarray Analysis / Q000379:methods*; D008826:Microbial Sensitivity Tests; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D012293:Rifampin / Q000494:pharmacology*; D018088:Tuberculosis, Multidrug-Resistant / Q000382:microbiology*
chemical_list: D000995:Antitubercular Agents; D007538:Isoniazid; D012293:Rifampin
keywords: Mycobacterium tuberculosis; biochip; drug resistance; enzymatic color reaction; mixed sample; templex PCR