Summary: Two novel pre-analytical sample treatment tools were evaluated in combination with four DNA extraction kits for the selective isolation of bacterial DNA from whole blood. Bacterial DNA extraction was performed after spiking whole blood with methicillin-resistant Staphylococcus aureus (MRSA) followed by amplification in a multiplex real-time PCR assay targeting three MRSA genes (mecA, femA and SA442). The combination of performing a pre-analytical sample treatment and using a larger sample volume increased the detection limit. MolYsis Complete5 was able to achieve bacterial detection at a concentration of 50 cfu per ml (corresponding to 4 cfu per PCR) and therefore achieved the lowest detection limit compared to all other DNA extraction methods. These results confirmed that the efficiency of DNA extraction, especially for clinical samples such as whole  blood, is a crucial element in the process of molecular pathogen detection. Ultimately, a combination of optimal sample processing and molecular detection techniques will lead to rapid and accurate pathogen detection for diagnosis of bloodstream infections.

 

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2) Horz HP, Scheer S, Vianna ME, Conrads G. 2009. New methods for selective isolation of bacterial DNA from human clinical specimens. Anaerobe doi:10.1016/j.anaerobe.2009.04.009 (in press)

 

Summary:  Separation of bacterial DNA from human DNA in clinical samples may have an important impact on downstream applications, involving microbial diagnostic systems. We evaluated two commercially available reagents (MolYsis, Molzym GmbH & Co. KG, Bremen and Pureprove®, SIRS-Lab GmbH, Jena, both Germany) for their potential to isolate and purify bacterial DNA from human DNA. We chose oral samples, which usually contain very high amounts of both human and bacterial cells. Three different DNA preparations each were made from eight caries- and eight periodontal specimens using the two reagents above and a conventional DNA extraction strategy as reference. Based on target-specific real-time-quantitative PCR assays we compared the reduction of human DNA versus loss of bacterial DNA. Human DNA was monitored by targeting the β-2-microglobulin gene, while bacteria were monitored by targeting 16S rDNA (total bacteria and Porphyromonas gingivalis) or the glycosyltransferase gene (Streptococcus mutans ). We found that in most cases at least 90% of human DNA could successfully be removed, with complete removal in eight of 16 cases using MolYsis, and two (of 16) cases using Pureprove. Conversely, detection of bacterial DNA was possible in all cases with a recovery rate generally ranging from 35% to 50%. In conclusion, both strategies have the potential to reduce background interference from the host DNA which may be of remarkable value for nucleic-acid based microbial diagnostic systems.