title: Triplex real-time PCR melting curve analysis for detecting Mycobacterium tuberculosis mutations associated with resistance to second-line drugs in a single reaction.
abstract: OBJECTIVE
  Resistance to anti-tuberculosis (TB) drugs has been a great challenge for global TB control. Limited tools have been developed to detect resistance to second-line drugs that are associated with extensively drug-resistant (XDR) TB. In this study we aimed to develop a simple and widely applicable assay for detecting mutations associated with second-line drug resistance in Mycobacterium tuberculosis.
  
  
  METHODS
  Three dually labelled probes targeting gyrA, rrs and the promoter of eis were designed to detect resistance to fluoroquinolones and second-line injectable agents (capreomycin, amikacin and kanamycin). A triplex reaction with all three probes and corresponding primers was first tested against 13 isolates with different mutations in the targeted regions. Then, the triplex assay was applied to 109 second-line drug-resistant isolates in a blind manner and the results were compared with the sequencing data.
  
  
  RESULTS
  All mutations in the targeted regions of 13 representative isolates could be detected through significant Tm reductions of the corresponding probe compared with the wild-type control. The detection results with 109 isolates were 100% concordant with sequencing data. Twelve ofloxacin-resistant isolates were detected as heteroresistant, indicating the coexistence of mutant and wild-type strains or the existence of different gyrA mutations.
  
  
  CONCLUSIONS
  We have developed a simple and widely applicable assay to detect second-line drug resistance of M. tuberculosis. This method, combined with assays for detecting first-line drug resistance, provides an efficient and reliable tool to diagnose multidrug-resistant TB and XDR-TB.
authors.affiliation: Key Laboratory of Medical Molecular Virology, Institutes of Biomedical Sciences and Institute of Medical Microbiology, Fudan University, Shanghai 200032, People's Republic of China.
authors.forename: Qingyun
authors.initials: Q
authors.lastname: Liu
authors.forename: Tao
authors.initials: T
authors.lastname: Luo
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Jian
authors.initials: J
authors.lastname: Mei
authors.forename: Qian
authors.initials: Q
authors.lastname: Gao
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D015345:Oligonucleotide Probes / Q000235:genetics; D017354:Point Mutation; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D044366:Transition Temperature
chemical_list: D000995:Antitubercular Agents; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes