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.
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References
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 41:580–637
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 61:1556–1562
Patel JB (2001) 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory. Mol Diagn 6:313–321
Woese CR (1987) Bacterial evolution. Microbiol Rev 51:221–271
Luna RA, Fasciano LR, Jones SC et al (2007) DNA pyrosequencing-based bacterial pathogen identification in a pediatric hospital setting. J Clin Microbiol 45:2985–2992
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: 1792–1795
Sundquist A, Bigdeli S, Jalili R et al (2007) Bacterial flora-typing with targeted, chip-based Pyrosequencing. BMC Microbiol 7:108
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: 5261–5267
Jordan JA, Jones-Laughner J, Durso MB (2009) Utility of pyrosequencing in identifying bacteria directly from positive blood culture bottles. J Clin Microbiol 47:368–372
Acknowledgments
We thank Maiko Motoshima and Sayaka Mori for technical assistance.
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Morinaga, Y., Yanagihara, K. (2015). Broad-Range PCR in the Identification of Bacterial and Fungal Pathogens from Positive Blood Culture Bottles: A Sequencing Approach. In: Mancini, N. (eds) Sepsis. Methods in Molecular Biology, vol 1237. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1776-1_7
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DOI: https://doi.org/10.1007/978-1-4939-1776-1_7
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