Related Experiment Video
Updated: Jun 11, 2026

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS
Published on: July 11, 2016
Simultaneous sequence analysis of the 16S rRNA and rpoB genes by use of RipSeq software to identify Mycobacterium
Keith E Simmon1, Øyvind Kommedal, Øystein Saebo
1Associated Regional and University Pathologists (ARUP) Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108, USA. keith.simmon@aruplab.com
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.
Related Concept Videos
Rapid Identification of Pathogens
Modern Molecular Taxonomy
Applications of Molecular Taxonomy
RNA-seq
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...
Ribosome Profiling
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique helps...

