title: Simultaneous sequence analysis of the 16S rRNA and rpoB genes by use of RipSeq software to identify Mycobacterium species.
abstract: The 16S rRNA gene is commonly used to identify Mycobacterium spp., but alternative DNA targets can provide better resolution to the species level. We evaluated a novel system that enables simultaneous amplification, sequencing, and analysis of two different DNA targets in a single tube to identify clinical isolates of Mycobacterium spp. For 139 clinical isolates, we found that the 16S rRNA gene alone identified 67 (48%) isolates as single species, 68 (49%) isolates to the complex or group level, and 4 (3%) isolates to the genus level only. The rpoB gene alone identified 117 (84%) isolates as single species, 10 (7%) isolates to the complex or group level, and 12 (8%) isolates to the genus level only. Combining the separate analyses for sequencing of two gene targets, 119 (86%) isolates were identified as single species and 10 (7%) isolates were identified to the complex or group level. Seven (5%) isolates were identified as novel species within established groups, and 3 (2%) were identified to the genus level only. Dual-locus identification identified 110 (79%) isolates as single species and 22 (16%) isolates to the complex or group level. Six (4%) were identified as novel species within established groups, and 1 (1%) was identified to the genus level only. Identifications were more accurate when both the 16S rRNA and rpoB genes were screened, and reliance on a single gene target was suboptimal. RipSeq dual-locus software provides an accurate alternative method for laboratories using two different gene targets for microorganism identification.
authors.affiliation: Associated Regional and University Pathologists (ARUP) Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108, USA. keith.simmon@aruplab.com
authors.forename: Keith E
authors.initials: KE
authors.lastname: Simmon
authors.forename: Øyvind
authors.initials: Ø
authors.lastname: Kommedal
authors.forename: Øystein
authors.initials: Ø
authors.lastname: Saebo
authors.forename: Bjarte
authors.initials: B
authors.lastname: Karlsen
authors.forename: Cathy A
authors.initials: CA
authors.lastname: Petti
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D006801:Humans; D009161:Mycobacterium / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014376:Tuberculosis / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit