Abstract
Molecular diagnostics is an increasing popular approach for the direct detection and identification of pathogenic bacteria in clinical samples. Conventional culture techniques are time-consuming and therefore causing a delay in the diagnosis of the patient. Alternative techniques based on nucleic acid amplification offer a shorter turn-around-time and the ability to identify fastidious and non-cultivable organisms. However, molecular detection of bacteria in blood, by for example PCR, RT-PCR, or sequencing of the 16S rDNA genes is often complicated by the presence of PCR-inhibitory compounds. Here we describe several different methods for the extraction of bacterial DNA from whole blood samples. The methods differ regarding costs, hands-on time as well as regarding sensitivity. In combination with a model PCR the detection limits that can be reached using the different methods range from 1,000 to 50 cfu/ml.
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References
Peters RP, van Agtmael MA, Danner SA, Savelkoul PH, Vandenbroucke-Grauls CM (2004) New developments in the diagnosis of bloodstream infections. Lancet Infect Dis 4:751–760
Centers for Disease Control and Prevention (1992) Advance report of final mortality statistics. Centers for Disease Control and Prevention. Mon Vital Stat Rep 40:1–13
Glerant JC, Hellmuth D, Schmit JL, Ducroix JP, Jounieaux V (1999) Utility of blood cultures in community-acquired pneumonia requiring hospitalization: influence of antibiotic treatment before admission. Respir Med 93:208–212
Grace CJ, Lieberman J, Pierce K, Littenberg B (2001) Usefulness of blood culture for hospitalized patients who are receiving antibiotic therapy. Clin Infect Dis 32:1651–1655
McKenzie R, Reimer LG (1987) Effect of antimicrobials on blood cultures in endocarditis. Diagn Microbiol Infect Dis 8:165–172
Jordan JA, Durso MB (2000) Comparison of 16S rRNA gene PCR and BACTEC 9240 for detection of neonatal bacteremia. J Clin Microbiol 38:2574–2578
Klaschik S, Lehmann LE, Raadts A et al (2004) Detection and differentiation of in vitro-spiked bacteria by real-time PCR and melting-curve analysis. J Clin Microbiol 42:512–517
Nadkarni MA, Martin FE, Jacques NA, Hunter N (2002) Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology 148:257–266
Rothman RE, Majmudar MD, Kelen GD et al (2002) Detection of bacteremia in emergency department patients at risk for infective endocarditis using universal 16S rRNA primers in a decontaminated polymerase chain reaction assay. J Infect Dis 186:1677–1681
Shafazand S, Weinacker AB (2002) Blood cultures in the critical care unit: improving utilization and yield. Chest 122:1727–1736
Kane TD, Alexander JW, Johannigman JA (1998) The detection of microbial DNA in the blood: a sensitive method for diagnosing bacteremia and/or bacterial translocation in surgical patients. Ann Surg 227:1–9
Klaschik S, Lehmann LE, Raadts A, Book M, Hoeft A, Stuber F (2002) Real-time PCR for detection and differentiation of gram-positive and gram-negative bacteria. J Clin Microbiol 40:4304–4307
Barken KB, Haagensen JA, Tolker-Nielsen T (2007) Advances in nucleic acid-based diagnostics of bacterial infections. Clin Chim Acta 384:1–11
Al-Soud WA, Radstrom P (2001) Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol 39:485–493
Kreader CA (1996) Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl Environ Microbiol 62:1102–1106
Morata P, Queipo-Ortuno MI, de Dios Colmenero J (1998) Strategy for optimizing DNA amplification in a peripheral blood PCR assay used for diagnosis of human brucellosis. J Clin Microbiol 36:2443–2446
Smith K, Diggle MA, Clarke SC (2003) Comparison of commercial DNA extraction kits for extraction of bacterial genomic DNA from whole-blood samples. J Clin Microbiol 41:2440–2443
Wilson IG (1997) Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63:3741–3751
Rantakokko-Jalava K, Jalava J (2002) Optimal DNA isolation method for detection of bacteria in clinical specimens by broad-range PCR. J Clin Microbiol 40:4211–4217
Zucol F, Ammann RA, Berger C et al (2006) Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification. J Clin Microbiol 44:2750–2759
Horz HP, Scheer S, Huenger F, Vianna ME, Conrads G (2008) Selective isolation of Âbacterial DNA from human clinical specimens. J Microbiol Methods 72:98–102
Hansen WL, Bruggeman CA, Wolffs PF (2009) Evaluation of new preanalysis sample treatment tools and DNA isolation protocols to improve bacterial pathogen detection in whole blood. J Clin Microbiol 47:2629–2631
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Hansen, W.L.J., Bruggeman, C.A., Wolffs, P.F.G. (2013). Pre-analytical Sample Treatment and DNA Extraction Protocols for the Detection of Bacterial Pathogens from Whole Blood. In: Wilks, M. (eds) PCR Detection of Microbial Pathogens. Methods in Molecular Biology, vol 943. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-353-4_4
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DOI: https://doi.org/10.1007/978-1-60327-353-4_4
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Publisher Name: Humana Press, Totowa, NJ
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