title: Detection of first-line anti-tuberculosis drug resistance mutations by allele-specific primer extension on a microsphere-based platform.
abstract: BACKGROUND
  Resistance of Mycobacterium tuberculosis to anti-tuberculosis (TB) drugs is almost exclusively due to spontaneous chromosomal mutations in target genes. Rapid detection of drug resistance to both first- and second-line anti-TB drugs has become a key component of TB control programs. Technologies that allow rapid, cost-effective, and high-throughput detection of specific nucleic acid sequences are needed. This study was to develop a high-throughput assay based on allele-specific primer extension (ASPE) and MagPlex-TAG microspheres to detect anti-TB drug resistance mutations.
  
  
  METHODS
  DNA samples from 357 M. tuberculosis clinical isolates and H37Rv were amplified by multiplex PCR using four primer sets, followed by multiplex ASPE using 23 TAG-ASPE primers. The products were sorted on the TAG-ASPE array and detected by using the Luminex xMAP system. Genotypes were also determined by sequencing.
  
  
  RESULTS
  Genetic drug susceptibility typing by the TAG-ASPE method was 100% concordant with those obtained by sequencing. Compared with phenotypic drug susceptibility testing (DST) as a reference method, the sensitivity and specificity of the TAG-ASPE method were 83% (95% confidence interval [CI], 79-88%) and 97% (95% CI, 90-100%) for isoniazid. For rifampin testing, the sensitivity and specificity were 90% (95% CI, 86-93%) and 100% (95% CI, 99-100%). Also, the sensitivity and specificity were 58% (95% CI, 51-65%) and 86% (95% CI, 79-93%) for ethambutol.
  
  
  CONCLUSIONS
  This study demonstrated the TAG-ASPE method is suitable for highly reproducible, cost-effective, and high-throughput clinical genotyping applications.
authors.affiliation: Korean Institute of Tuberculosis, Cheongju, Korea. seung6992@hanmail.net.
authors.forename: Seung Heon
authors.initials: SH
authors.lastname: Lee
authors.affiliation: Genes Laboratories, Seongnam, Korea.
authors.forename: Hee Baeg
authors.initials: HB
authors.lastname: Choi
authors.affiliation: Department of Pathology, Semyung University, Jecheon, Korea.
authors.forename: Sung Yul
authors.initials: SY
authors.lastname: Yu
authors.affiliation: Genes Laboratories, Seongnam, Korea.
authors.forename: Uck Jin
authors.initials: UJ
authors.lastname: Chang
authors.affiliation: Korean Institute of Tuberculosis, Cheongju, Korea.
authors.forename: Chang Ki
authors.initials: CK
authors.lastname: Kim
authors.affiliation: Korean Institute of Tuberculosis, Cheongju, Korea.
authors.forename: Hee Jin
authors.initials: HJ
authors.lastname: Kim
mesh_terms: D000483:Alleles; D000995:Antitubercular Agents / Q000494:pharmacology*; D001483:Base Sequence; D017931:DNA Primers / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024901:Drug Resistance, Multiple, Bacterial; D005838:Genotype; D006801:Humans; D008826:Microbial Sensitivity Tests; D008863:Microspheres; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D017931:DNA Primers; D004269:DNA, Bacterial
keywords: Allele-specific primer extension; Drug-resistance; Genotyping; Mycobacteirum tuberculosis