title: Detection and identification of bacteria in clinical samples by 16S rRNA gene sequencing: comparison of two different approaches in clinical practice.
abstract: Amplification and sequence analysis of the 16S rRNA gene can be applied to detect and identify bacteria in clinical samples. We examined 75 clinical samples (17 culture-positive, 58 culture-negative) prospectively by two different PCR protocols, amplifying either a single fragment (1343 bp) or two fragments (762/598 bp) of the 16S rRNA gene. The 1343 bp PCR and 762/598 bp PCRs detected and identified the bacterial 16S rRNA gene in 23 (31 %) and 38 (51 %) of the 75 samples, respectively. The 1343 bp PCR identified 19 of 23 (83 %) PCR-positive samples to species level while the 762/598 bp PCR identified 14 of 38 (37 %) bacterial 16S rRNA gene fragments to species level and 24 to the genus level only. Amplification of shorter fragments of the bacterial 16S rRNA gene (762 and 598 bp) resulted in a more sensitive assay; however, analysis of a large fragment (1343 bp) improved species discrimination. Although not statistically significant, the 762/598 bp PCR detected the bacterial 16S rRNA gene in more samples than the 1343 bp PCR, making it more likely to be a more suitable method for the primary detection of the bacterial 16S rRNA gene in the clinical setting. The 1343 bp PCR may be used in combination with the 762/598 bp PCR when identification of the bacterial rRNA gene to species level is required.
authors.affiliation: Colindale Microbiological Services, Health Protection Agency, Colindale, London NW9 5AT, UK.|Department of Medical Microbiology, Royal Free Hospital NHS Trust, Pond Street, London NW3 2PF, UK.
authors.forename: Claire
authors.initials: C
authors.lastname: Jenkins
authors.affiliation: Colindale Microbiological Services, Health Protection Agency, Colindale, London NW9 5AT, UK.|Department of Medical Microbiology, Royal Free Hospital NHS Trust, Pond Street, London NW3 2PF, UK.
authors.forename: Clare L
authors.initials: CL
authors.lastname: Ling
authors.affiliation: Centre for Clinical Microbiology, Department of Infection, Royal Free Campus, UCL, London NW3 2QG, UK.|Department of Medical Microbiology, Royal Free Hospital NHS Trust, Pond Street, London NW3 2PF, UK.
authors.forename: Holly L
authors.initials: HL
authors.lastname: Ciesielczuk
authors.affiliation: Department of Medical Microbiology, Royal Free Hospital NHS Trust, Pond Street, London NW3 2PF, UK.
authors.forename: Julianne
authors.initials: J
authors.lastname: Lockwood
authors.affiliation: Centre for Clinical Microbiology, Department of Infection, Royal Free Campus, UCL, London NW3 2QG, UK.|Department of Medical Microbiology, Royal Free Hospital NHS Trust, Pond Street, London NW3 2PF, UK.
authors.forename: Susan
authors.initials: S
authors.lastname: Hopkins
authors.affiliation: Centre for Clinical Microbiology, Department of Infection, Royal Free Campus, UCL, London NW3 2QG, UK.
authors.forename: Timothy D
authors.initials: TD
authors.lastname: McHugh
authors.affiliation: School of Medicine, University of St Andrews, North Haugh, St Andrews, Fife KY16 9TF, UK.|Centre for Clinical Microbiology, Department of Infection, Royal Free Campus, UCL, London NW3 2QG, UK.
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Gillespie
authors.affiliation: Department of Medical Microbiology, Royal Free Hospital NHS Trust, Pond Street, London NW3 2PF, UK.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Kibbler
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000235:genetics*; D001424:Bacterial Infections / Q000175:diagnosis / Q000382:microbiology*; D001431:Bacteriological Techniques / Q000379:methods; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry; D006801:Humans; D010996:Pleural Effusion / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013492:Suppuration / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S