title: Characterization of polybacterial clinical samples using a set of group-specific broad-range primers targeting the 16S rRNA gene followed by DNA sequencing and RipSeq analysis.
abstract: The standard use of a single universal broad-range PCR in direct 16S rDNA sequencing from polybacterial samples leaves the minor constituents at risk of remaining undetected because all bacterial DNA will be competing for the same reagents. In this article we introduce a set of three broad-range group-specific 16S rDNA PCRs that together cover the clinically relevant bacteria and apply them in the investigation of 25 polybacterial clinical samples. Mixed DNA chromatograms from samples containing more than one species per primer group were analysed using RipSeq Mixed (iSentio, Norway), a web-based application for the interpretation of chromatograms containing up to three different species. The group-specific PCRs reduced complexity in the resulting DNA chromatograms and made the assay more sensitive in situations with unequal species concentrations. Together this allowed for identification of a significantly higher number of bacterial species than did standard direct sequencing with a single universal primer pair and RipSeq analysis (95 vs 51). The method could improve microbiological diagnostics for important groups of patients and can be established in any laboratory with experience in direct 16S rDNA sequencing.
authors: Section for Microbiology and Immunology, The Gade Institute, University of Bergen, 5020 Bergen, Norway.
authors: Øyvind
authors: Ø
authors: Kommedal
authors: Institute of Biology, University of Bergen, 5020 Bergen, Norway.
authors: Katrine
authors: K
authors: Lekang
authors: Institute of Medicine, University of Bergen, 5020 Bergen, Norway.
authors: Nina
authors: N
authors: Langeland
authors: Department of Microbiology, Haukeland University Hospital, 5021 Bergen, Norway.|Section for Microbiology and Immunology, The Gade Institute, University of Bergen, 5020 Bergen, Norway.
authors: Harald G
authors: HG
authors: Wiker
mesh_terms: D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification*; D001424:Bacterial Infections / Q000382:microbiology; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D015203:Reproducibility of Results; D021161:Self-Sustained Sequence Replication / Q000379:methods*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S