1887

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.

  • This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.028373-0
2011-07-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jmm/60/7/927.html?itemId=/content/journal/jmm/10.1099/jmm.0.028373-0&mimeType=html&fmt=ahah

References

  1. Al Masalma M., Armougom F., Scheld W. M., Dufour H., Roche P.-H., Drancourt M., Raoult D. 2009; The expansion of the microbiological spectrum of brain abscesses with use of multiple 16S ribosomal DNA sequencing. Clin Infect Dis 48:1169–1178 [View Article][PubMed]
    [Google Scholar]
  2. Altman D. G. 1991; Non-parametric analysis. In Practical Statistics for Medical Research, 1st edn. pp. 203–205 London: Chapman & Hall;
    [Google Scholar]
  3. Baker G. C., Smith J. J., Cowan D. A. 2003; Review and re-analysis of domain-specific 16S primers. J Microbiol Methods 55:541–555 [View Article][PubMed]
    [Google Scholar]
  4. Bosshard P. P., Zbinden R., Altwegg M. 2002; Turicibacter sanguinis gen. nov., sp. nov., a novel anaerobic, Gram-positive bacterium. Int J Syst Evol Microbiol 52:1263–1266 [View Article][PubMed]
    [Google Scholar]
  5. Bosshard P. P., Abels S., Zbinden R., Böttger E. C., Altwegg M. 2003; Ribosomal DNA sequencing for identification of aerobic gram-positive rods in the clinical laboratory (an 18-month evaluation). J Clin Microbiol 41:4134–4140 [View Article][PubMed]
    [Google Scholar]
  6. Bosshard P. P., Abels S., Altwegg M., Böttger E. C., Zbinden R. 2004; Comparison of conventional and molecular methods for identification of aerobic catalase-negative gram-positive cocci in the clinical laboratory. J Clin Microbiol 42:2065–2073 [View Article][PubMed]
    [Google Scholar]
  7. de Vries J. J., Arents N. L., Manson W. L. 2008; Campylobacter species isolated from extra-oro-intestinal abscesses: a report of four cases and literature review. Eur J Clin Microbiol Infect Dis 27:1119–1123 [View Article][PubMed]
    [Google Scholar]
  8. Di Giusto D. A., King G. C. 2004; Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: implications for genotyping assays. Nucleic Acids Res 32:e32 [View Article][PubMed]
    [Google Scholar]
  9. Edwards U., Rogall T., Blöcker H., Emde M., Böttger E. C. 1989; Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17:7843–7853 [View Article][PubMed]
    [Google Scholar]
  10. Goldenberger D., Künzli A., Vogt P., Zbinden R., Altwegg M. 1997; Molecular diagnosis of bacterial endocarditis by broad-range PCR amplification and direct sequencing. J Clin Microbiol 35:2733–2739[PubMed]
    [Google Scholar]
  11. Harris K. A., Hartley J. C. 2003; Development of broad-range 16S rDNA PCR for use in the routine diagnostic clinical microbiology service. J Med Microbiol 52:685–691 [View Article][PubMed]
    [Google Scholar]
  12. Hartmeyer G. N., Justesen U. S. 2010; Direct 16S rRNA gene sequencing of polymicrobial culture-negative samples with analysis of mixed chromatograms. J Med Microbiol 59:486–488 [View Article][PubMed]
    [Google Scholar]
  13. Johnson C. C., Reinhardt J. F., Edelstein M. A. C., Mulligan M. E., George W. L., Finegold S. M. 1985; Bacteroides gracilis, an important anaerobic bacterial pathogen. J Clin Microbiol 22:799–802[PubMed]
    [Google Scholar]
  14. Kommedal Ø., Karlsen B., Saebø Ø. 2008; Analysis of mixed sequencing chromatograms and its application in direct 16S rRNA gene sequencing of polymicrobial samples. J Clin Microbiol 46:3766–3771 [View Article][PubMed]
    [Google Scholar]
  15. Kommedal Ø., Kvello K., Skjåstad R., Langeland N., Wiker H. G. 2009; Direct 16S rRNA gene sequencing from clinical specimens, with special focus on polybacterial samples and interpretation of mixed DNA chromatograms. J Clin Microbiol 47:3562–3568 [View Article][PubMed]
    [Google Scholar]
  16. Petti C. A., Bosshard P. P., Brandt M. E., Clarridge J. E. III, Feldblyum T. V., Foxall P., Furtado M. R., Pace N., Procop G. 2008a Interpretive Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing Approved Guideline Document MM18-A, 28 Wayne, PA: Clinical and Laboratory Standards Institute;
    [Google Scholar]
  17. Petti C. A., Simmon K. E., Bender J., Blaschke A., Webster K. A., Conneely M. F., Schreckenberger P. C., Origitano T. C., Challapalli M. 2008b). Culture-negative intracerebral abscesses in children and adolescents from Streptococcus anginosus group infection: a case series. Clin Infect Dis 46:1578–1580 [View Article][PubMed]
    [Google Scholar]
  18. Rupp J., Solbach W., Gieffers J. 2006; Single-nucleotide-polymorphism-specific PCR for quantification and discrimination of Chlamydia pneumoniae genotypes by use of a “locked” nucleic acid. Appl Environ Microbiol 72:3785–3787 [View Article][PubMed]
    [Google Scholar]
  19. Schuurman T., de Boer R. F., Kooistra-Smid A. M. D., van Zwet A. A. 2004; Prospective study of use of PCR amplification and sequencing of 16S ribosomal DNA from cerebrospinal fluid for diagnosis of bacterial meningitis in a clinical setting. J Clin Microbiol 42:734–740 [View Article][PubMed]
    [Google Scholar]
  20. Senn L., Franciolli M., Raoult D., Moulin A., Von Segesser L., Calandra T., Greub G. 2005; Coxiella burnetii vascular graft infection. BMC Infect Dis 5:109 [View Article][PubMed]
    [Google Scholar]
  21. Wellinghausen N., Kochem A.-J., Disqué C., Mühl H., Gebert S., Winter J., Matten J., Sakka S. G. 2009; Diagnosis of bacteremia in whole-blood samples by use of a commercial universal 16S rRNA gene-based PCR and sequence analysis. J Clin Microbiol 47:2759–2765 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.028373-0
Loading
/content/journal/jmm/10.1099/jmm.0.028373-0
Loading

Data & Media loading...

Supplements

Supplementary File 1

Supplementary File 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error