title: Integrated microfluidic card with TaqMan probes and high-resolution melt analysis to detect tuberculosis drug resistance mutations across 10 genes.
abstract: Genotypic methods for drug susceptibility testing of Mycobacterium tuberculosis are desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We developed a microfluidic TaqMan array card (TAC) that utilizes both sequence-specific probes and high-resolution melt analysis (HRM), providing two layers of detection of mutations. Twenty-seven primer pairs and 40 probes were designed to interrogate 3,200 base pairs of critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncA genes. The method was evaluated on 230 clinical M. tuberculosis isolates from around the world, and it yielded 96.1% accuracy (2,431/2,530) in comparison to that of Sanger sequencing and 87% accuracy in comparison to that of the slow culture-based susceptibility testing. This TAC-HRM method integrates assays for 10 genes to yield fast, comprehensive, and accurate drug susceptibility results for the 9 major antibiotics used to treat TB and could be deployed to improve treatment outcomes.
  
  
  OBJECTIVE
  Multidrug-resistant tuberculosis threatens global tuberculosis control efforts. Optimal therapy utilizes susceptibility test results to guide individualized treatment regimens; however, the susceptibility testing methods in use are technically difficult and slow. We developed an integrated TaqMan array card method with high-resolution melt analysis that interrogates 10 genes to yield a fast, comprehensive, and accurate drug susceptibility result for the 9 major antituberculosis antibiotics.
authors.affiliation: Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Suporn
authors.initials: S
authors.lastname: Pholwat
authors.affiliation: Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.affiliation: Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Stroup
authors.affiliation: Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Jean
authors.initials: J
authors.lastname: Gratz
authors.affiliation: International Center for Diarrheal Diseases and Research, Dhaka, Bangladesh.
authors.forename: Sayera
authors.initials: S
authors.lastname: Banu
authors.affiliation: International Center for Diarrheal Diseases and Research, Dhaka, Bangladesh.
authors.forename: S M Mazidur
authors.initials: SM
authors.lastname: Rahman
authors.affiliation: International Center for Diarrheal Diseases and Research, Dhaka, Bangladesh.
authors.forename: Sara Sabrina
authors.initials: SS
authors.lastname: Ferdous
authors.affiliation: Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Suporn
authors.initials: S
authors.lastname: Foongladda
authors.affiliation: Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Duangjai
authors.initials: D
authors.lastname: Boonlert
authors.forename: Oleg
authors.initials: O
authors.lastname: Ogarkov
authors.affiliation: Scientific Center of Family Health and Reproductive Problems, Siberian Branch, Russian Academy of Medical Sciences, Irkutsk, Russia.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Zhdanova
authors.affiliation: Kilimanjaro Clinical Research Institute, Moshi, Tanzania.
authors.forename: Gibson
authors.initials: G
authors.lastname: Kibiki
authors.affiliation: Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Heysell
authors.affiliation: Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia, USA erh6k@virginia.edu.
authors.forename: Eric
authors.initials: E
authors.lastname: Houpt
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D060005:Genotyping Techniques / Q000295:instrumentation / Q000379:methods*; D056656:Lab-On-A-Chip Devices; D008826:Microbial Sensitivity Tests / Q000295:instrumentation / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D044366:Transition Temperature
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes