Detection and identification of bacteria in clinical samples by 16S rRNA gene sequencing: comparison of two different approaches in clinical practice

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 f...

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Bibliographic Details
Published in:	Journal of medical microbiology Vol. 61; no. 4; pp. 483 - 488
Main Authors:	JENKINS, Claire, LING, Clare L, CIESIELCZUK, Holly L, LOCKWOOD, Julianne, HOPKINS, Susan, MCHUGH, Timothy D, GILLESPIE, Stephen H, KIBBLER, Christopher C
Format:	Journal Article
Language:	English
Published:	Reading Society for General Microbiology 01-04-2012
Subjects:	Bacteria - classification Bacteria - genetics Bacterial Infections - diagnosis Bacterial Infections - microbiology Bacteriological Techniques - methods Bacteriology Base Sequence Biological and medical sciences DNA, Bacterial - chemistry DNA, Bacterial - genetics DNA, Ribosomal - chemistry Fundamental and applied biological sciences. Psychology Humans Infectious diseases Medical sciences Microbiology Miscellaneous Pleural Effusion - microbiology Polymerase Chain Reaction - methods RNA, Bacterial - genetics RNA, Ribosomal, 16S - genetics Sequence Alignment Sequence Analysis, DNA Suppuration - microbiology 16S-RNA Bacteria Identification Detection Ribosomal DNA
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Summary:	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.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0022-2615 1473-5644
DOI:	10.1099/jmm.0.030387-0