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. 2007 May;69(2):330-9.
doi: 10.1016/j.mimet.2007.02.005. Epub 2007 Feb 22.

A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria

Soumitesh Chakravorty  1 , Danica Helb, Michele Burday, Nancy Connell, David Alland
Affiliations

A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria

Soumitesh Chakravorty et al. J Microbiol Methods. 2007 May.

Abstract

Bacterial 16S ribosomal RNA (rRNA) genes contain nine "hypervariable regions" (V1-V9) that demonstrate considerable sequence diversity among different bacteria. Species-specific sequences within a given hypervariable region constitute useful targets for diagnostic assays and other scientific investigations. No single region can differentiate among all bacteria; therefore, systematic studies that compare the relative advantage of each region for specific diagnostic goals are needed. We characterized V1-V8 in 110 different bacterial species including common blood borne pathogens, CDC-defined select agents and environmental microflora. Sequence similarity dendrograms were created for hypervariable regions V1-V8, and for selected combinations of regions or short segments within individual hypervariable regions that might be appropriate for DNA probing and real-time PCR. We determined that V1 best differentiated among Staphylococcus aureus and coagulase negative Staphylococcus sp. V2 and V3 were most suitable for distinguishing all bacterial species to the genus level except for closely related enterobacteriaceae. V2 best distinguished among Mycobacterium species and V3 among Haemophilus species. The 58 nucleotides-long V6 could distinguish among most bacterial species except enterobacteriaceae. V6 was also noteworthy for being able to differentiate among all CDC-defined select agents including Bacillus anthracis, which differed from B. cereus by a single polymorphism. V4, V5, V7 and V8 were less useful targets for genus or species-specific probes. The hypervariable sequence-specific dendrograms and the "MEGALIGN" files provided online will be highly useful tools for designing specific probes and primers for molecular assays to detect pathogenic bacteria, including select agents.

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Figures

Fig.1
Fig.1
Dendrograms of sequence alignments of 16S rRNA gene hypervariable segments of the bacterial species included in this study.a:V3; b:V6; c:a concatenated sequence of V2, V3 and V6.
Fig.1
Fig.1
Dendrograms of sequence alignments of 16S rRNA gene hypervariable segments of the bacterial species included in this study.a:V3; b:V6; c:a concatenated sequence of V2, V3 and V6.
Fig.1
Fig.1
Dendrograms of sequence alignments of 16S rRNA gene hypervariable segments of the bacterial species included in this study.a:V3; b:V6; c:a concatenated sequence of V2, V3 and V6.
Fig. 2
Fig. 2
PCR amplification of 16S rRNA hypervariable regions of from 30 bacterial species using universal primers. Lane M: 100 kb molecular weight DNA marker; Lanes 1-30: Aerococcus viridans, Arcanobacterium pyogenes; Clostricium difficile; Bacillus anthracis, Bacteroides uniformis, Campylobacter jejuni, Burkholderia mallei, Corynebacterium pseudodiptheriticum, Enterococcus galinarium, Escherichia coli O157:H7, Francisella tularensis, Klebsiella pneumoniae, Haemophilus influenzae, Haemophilus parahemolyticus, Listeria grayi, Moraxella cattarhalis, Mycobacterium tuberculosis, Oligella urethralis, Proteus vulgaris, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Neisseria lactamica, Neisseria mucosa, Serratia marcescens, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis and Yersinia pestis. A: universal primers amplifying V3; B: universal primers amplifying V6.
Fig.3
Fig.3
Real time PCR plot showing amplification of S. aureus and S. epidermidis genomic DNA using universal primers to V6 and a Staphylococcus epidermidis specific molecular beacon. The number of bacterial genomes added to each PCR reaction are indicated. formula image 106 genomes formula image 105 genomes formula image 104 genomes formula image 103 genomes formula image 100 genomes formula image 10 genomes formula image 1 genome ― DNA negative control formula imageS. aureus 106 genomes
See this image and copyright information in PMC

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