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Broad-range Amplification and Sequencing of the rpoB Gene: A Novel Assay for Bacterial Identification in Clinical Microbiology

ABSTRACT: Broad-range Amplification and Sequencing of the rpoB Gene

PROVIDER: PRJEB75571 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR13093710_1.fastq.gz	Fastqsanger.gz
	ERR13093710_2.fastq.gz	Fastqsanger.gz
	ERR13093711_1.fastq.gz	Fastqsanger.gz
	ERR13093711_2.fastq.gz	Fastqsanger.gz
	ERR13093713_1.fastq.gz	Fastqsanger.gz

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Broad-range amplification and sequencing of the <i>rpoB</i> gene: a novel assay for bacterial identification in clinical microbiology.

Project description: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 the 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 specimens (88%). Broad-range rpoB amplification and sequencing provides a promising tool for bacterial identification, improving discrimination between closely related species and making it amenable for use in culture-based and culture-independent diagnostic approaches.

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