Sepsis pp 129-138 | Cite as

Broad-Range PCR for the Identification of Bacterial and Fungal Pathogens from Blood: A Sequencing Approach

  • Eva Leitner
  • Harald H. KesslerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1237)


Broad-range PCR has become a valuable tool for the identification of microorganisms in the clinical laboratory over the last years. It was primarily used to identify slow-growing and fastidious microorganisms with poor biochemical activity. Nowadays, it is also used to identify microorganisms directly from clinical samples such as blood or punctuates from primarily sterile body sites. In these specimens, the usage of broad-range PCR is challenging regarding contamination and standardization. To overcome these problems, a new test system, the SepsiTest™, was introduced recently employing broad-range PCR for the identification of microorganisms in septic patients. In this chapter, the test system is described and the equipment necessary listed.

Key words

Sepsis Broad-range PCR Sequencing SepsiTest Identification Bacteria Fungi 


  1. 1.
    Clarridge JE 3rd (2004) Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin Microbiol Rev 17:840–862, table of contentsPubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    Sontakke S, Cadenas MB, Maggi RG, Diniz PP, Breitschwerdt EB (2009) Use of broad range16S rDNA PCR in clinical microbiology. J Microbiol Methods 76:217–225PubMedCrossRefGoogle Scholar
  3. 3.
    Atkins SD, Clark IM (2004) Fungal molecular diagnostics: a mini review. J Appl Genet 45:3–15PubMedGoogle Scholar
  4. 4.
    Klappenbach JA, Saxman PR, Cole JR, Schmidt TM (2001) rrndb: the ribosomal RNA operon copy number database. Nucleic Acids Res 29:181–184PubMedCrossRefPubMedCentralGoogle Scholar
  5. 5.
    CLSI, Clinical and Laboratory Standards Institute (2008) Interpretive criteria for identification of bacteria and fungi by DNA target sequencing; approved guideline. CLSI dokument MM18-A (ISBN 1-56238-664-6)Google Scholar
  6. 6.
    Wellinghausen N, Kochem AJ, Disque C, Muhl H, Gebert S, Winter J, Matten J, Sakka SG (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 47:2759–2765PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Leitner E, Kessler HH, Spindelboeck W, Hoenigl M, Putz-Bankuti C, Stadlbauer-Kollner V, Krause R, Grisold AJ, Feierl G, Stauber RE (2013) Comparison of two molecular assays with conventional blood culture for diagnosis of sepsis. J Microbiol Methods 92:253–255PubMedCrossRefGoogle Scholar
  8. 8.
    Wiesinger-Mayr H, Jordana-Lluch E, Martro E, Schoenthaler S, Noehammer C (2011) Establishment of a semi-automated pathogen DNA isolation from whole blood and comparison with commercially available kits. J Microbiol Methods 85:206–213PubMedCrossRefGoogle Scholar
  9. 9.
    Sleigh J, Cursons R, La Pine M (2001) Detection of bacteraemia in critically ill patients using 16S rDNA polymerase chain reaction and DNA sequencing. Intensive Care Med 27:1269–1273PubMedCrossRefGoogle Scholar
  10. 10.
    Fenollar F, Raoult D (2007) Molecular diagnosis of bloodstream infections caused by non-cultivable bacteria. Int J Antimicrob Agents 30(Suppl 1):S7–S15PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Institute of Hygiene, Microbiology and Environmental Medicine (IHME)Medical University of GrazGrazAustria

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