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Environmental Analysis

Bacterial DNA barcode sequence identification [M220]

Published: July 8th, 2009

Revised: February 25th, 2015

This test provides a low-level identification of a single bacterial isolate by sequencing of portions of the 16S ribosomal small subunit gene region. DNA-based sequence identification is the gold-standard for microbial identification, since it is capable of identifying rare or atypical isolates of bacteria irrespective of their responses to diagnostic culture media or biochemical tests.

In this test, we carefully cultivate the bacterial isolate in a broth medium, typically Tryptic Soy Broth (TSB). Log-phase cultures are harvested by centrifugation and DNA is isolated from cells using standard procedures. A diagnostic portion of the ribosomal small subunit gene is amplified by PCR and sequenced in both directions. A consensus sequence of both forward and reverse strands is constructed, and this sequence is used to query the Ribosomal Database Project (RDP), an online data analysis and alignment tool consisting of annotated Bacterial and Archaeal small-subunit 16S rRNA sequences. This approach to bacteria identification is unmatched in its ability to accurately identify troublesome isolates, or to ascertain the taxonomic position of novel strains.

Results of this test are reported as an identification or series of putative identifications with corresponding confidence measures. A phylogram generated using the UPGMA algorithm indicating the phylogenetic placement of the isolate relative to closely related taxa will is also provided.

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