Simultaneous sequence analysis of the 16S rRNA and rpoB genes by use of RipSeq software to identify Mycobacterium species.

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.