title: Dual priming oligonucleotides for broad-range amplification of the bacterial 16S rRNA gene directly from human clinical specimens.
abstract: Broad-range amplification and sequencing of the bacterial 16S rRNA gene directly from clinical specimens are offered as a diagnostic service in many laboratories. One major pitfall is primer cross-reactivity with human DNA which will result in mixed chromatograms. Mixed chromatograms will complicate subsequent sequence analysis and impede identification. In SYBR green real-time PCR assays, it can also affect crossing threshold values and consequently the status of a specimen as positive or negative. We evaluated two conventional primer pairs in common use and a new primer pair based on the dual priming oligonucleotide (DPO) principle. Cross-reactivity was observed when both conventional primer pairs were used, resulting in interpretation difficulties. No cross-reactivity was observed using the DPOs even in specimens with a high ratio of human to bacterial DNA. In addition to reducing cross-reactivity, the DPO principle also offers a high degree of flexibility in the design of primers and should be considered for any PCR assay intended for detection and identification of pathogens directly from human clinical specimens.
authors.affiliation: Section for Microbiology and Immunology, the Gade Institute, University of Bergen, Bergen, Norway. oyvind.kommedal@isentio.com
authors.forename: Øyvind
authors.initials: Ø
authors.lastname: Kommedal
authors.forename: Keith
authors.initials: K
authors.lastname: Simmon
authors.forename: Dilek
authors.initials: D
authors.lastname: Karaca
authors.forename: Nina
authors.initials: N
authors.lastname: Langeland
authors.forename: Harald G
authors.initials: HG
authors.lastname: Wiker
mesh_terms: D001419:Bacteria / Q000235:genetics*; D001424:Bacterial Infections / Q000175:diagnosis* / Q000382:microbiology; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics*; D006801:Humans; D025202:Molecular Diagnostic Techniques; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D059629:Signal-To-Noise Ratio
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S