Advertisement

Sepsis pp 65-72 | Cite as

Broad-Range PCR in the Identification of Bacterial and Fungal Pathogens from Positive Blood Culture Bottles: A Sequencing Approach

  • Yoshitomo Morinaga
  • Katsunori YanagiharaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1237)

Abstract

Rapid identification of causative bacteria in patients with sepsis can contribute to appropriate selection of antibiotics and improvement of patients’ prognosis. Genotypic identification is an emerging technology that may provide an alternative method to, or complement, established phenotypic identification procedures.

Sequence analysis of the 16S rRNA gene is a widely accepted tool for molecular identification of bacteria. Pyrosequencing is a DNA sequencing technique that is based on the detection of pyrophosphate that is released during DNA synthesis. Pyrosequencing can provide sequence information rapidly by reading short sequences; therefore, it may contribute to a rapid identification and lead to a great help in improving the outcome of sepsis. The DNA pyrosequencing-based identification from positive blood culture samples basically consisted of the following four steps: (1) DNA extraction, (2) amplification of target genes, (3) DNA pyrosequencing, and (4) homology searching.

Key words

Pyrosequence Genetic identification 16S rRNA V1 V3 Sepsis 

Notes

Acknowledgments

We thank Maiko Motoshima and Sayaka Mori for technical assistance.

References

  1. 1.
    Dellinger RP, Levy MM, Rhodes A et al (2013) Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012. Crit Care Med 41:580–637PubMedCrossRefGoogle Scholar
  2. 2.
    Motoshima M, Yanagihara K, Morinaga Y et al (2012) Identification of bacteria directly from positive blood culture samples by DNA pyrosequencing of the 16S rRNAgene. J Med Microbiol 61:1556–1562PubMedCrossRefGoogle Scholar
  3. 3.
    Patel JB (2001) 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory. Mol Diagn 6:313–321PubMedCrossRefGoogle Scholar
  4. 4.
    Woese CR (1987) Bacterial evolution. Microbiol Rev 51:221–271PubMedPubMedCentralGoogle Scholar
  5. 5.
    Luna RA, Fasciano LR, Jones SC et al (2007) DNA pyrosequencing-based bacterial pathogen identification in a pediatric hospital setting. J Clin Microbiol 45:2985–2992PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Park KS, Ki CS, Kang CI et al (2012) Evaluation of the GenBank, EzTaxon, and BIBI services for molecular identification of clinical blood culture isolates that were unidentifiable or misidentified by conventional methods. J Clin Microbiol 50: 1792–1795PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Sundquist A, Bigdeli S, Jalili R et al (2007) Bacterial flora-typing with targeted, chip-based Pyrosequencing. BMC Microbiol 7:108PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Wang Q, Garrity GM, Tiedje JM et al (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73: 5261–5267PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Jordan JA, Jones-Laughner J, Durso MB (2009) Utility of pyrosequencing in identifying bacteria directly from positive blood culture bottles. J Clin Microbiol 47:368–372PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Laboratory MedicineNagasaki University Graduate School of Biomedical SciencesNagasakiJapan

Personalised recommendations