Broad-range Amplification and Sequencing of the rpoB Gene

The rpoB gene has been proposed as a promising phylogenetic marker for bacterial identification, providing theoretically improved species-level resolution compared to the 16S rRNA gene for a range of clinically important taxa. However, its utility in diagnostic microbiology has been limited by the lack of broad-range primers allowing for its amplification from most species with a single PCR assay. Here, we present an assay for broad-range partial amplification and Sanger sequencing of the rpoB gene. To reduce cross-reactivity and allow for rpoB amplification directly from patient samples, primers were based on the dual priming oligonucleotide principle. The resulting amplicon is ~550 base pairs in length, and appropriate for species-level identification. Systematic in silico evaluation of a wide selection of taxa demonstrated improved resolution within multiple important genera, including, Enterococcus, Fusobacterium, Mycobacterium, Streptococcus, and Staphylococcus species, and several genera within the Enterobacteriaceae family. Broad-range rpoB amplification and Sanger sequencing of 115 bacterial isolates provided unambiguous species-level identification for 97 (84%) isolates, as compared to 57 (50%) using a clinical 16S rRNA gene assay. Several unresolved taxonomic matters disguised by low resolution of the 16S rRNA gene were revealed using the rpoB gene. Using a collection of 33 clinical specimens harboring bacteria and assumed to contain high concentrations of human DNA, the rpoB assay identified the pathogen in 29 (88%). Broad-range rpoB amplification and sequencing provides a promising tool for bacterial identification, improving discrimination between closely related species, making it amenable for use in culture-based and culture-independent diagnostic approaches.