Ribosomal DNA (16S rDNA) sequencing


Ribosomal DNA (16S rDNA) sequencing for organism identification

Collect and freeze (–70°C) unidentifiable culture isolate, tissue, or body fluid.


Physiologic Basis

16S ribosomal RNA (rRNA) gene sequencing is the current gold standard in bacterial and other pathogenic microorganism identification and classification.

Tests use universal PCR primers that bind to conserved regions of microbial rRNA (16s, and 18s/26s) genes and amplify adjacent hypervariable segments that are species-specific.


Indicated when: Culture-negative sample is suspected (eg, prior antibiotics, potential for atypical or rare organisms); establishing a definitive microbiologic diagnosis will change long-term antimicrobial plan (eg, culture-negative infective endocarditis, culture-negative prosthetic joint infection)


Identification of bacteria in clinical microbiology laboratories is typically performed by phenotypic tests such as Gram stain and biochemical tests, along with culture requirements and growth characteristics. These methods have limitations, eg, they can not identify bacteria with unusual or rare phenotypic profiles, slow-growing bacteria, uncultivable bacteria, and culture-negative infections. In addition, phenotypic tests may not be available for many newly described pathogenic microorganisms. In these clinical situations, 16S rDNA sequencing can help establish a definitive microbiologic diagnosis, that is, to rule in (with positive identification) or rule out an infection.

Tests are available for identification of bacterial, fungal and mycobacterial organisms.

Lasken RS et al. Recent advances in genomic DNA sequencing of microbial species from single cells. Nat Rev Genet 2014;15:577.  [PMID: 25091868]

Lluch J et al. The characterization of novel tissue microbiota using an optimized 16S metagenomic sequencing pipeline. PLoS One 2015;10:e0142334.  [PMID: 26544956]

Tremblay J et al. Primer and platform effects on 16S rRNA tag sequencing. Front Microbiol 2015;6:771.  [PMID: 26300854]