One Protocol for Preparation of Bacterial and Fungal DNA

Systemic infections and infections of other primary sterile body sites are caused by a great variety of microorganisms. A recent compilation of results grouped 200 isolates from wound, sepsis and other infections in intensive care units to Gram-positive cocci, Gram-negative aerobic rods, Gram-negative facultative rods, anaerobes and fungi [1].

For more than 10 years, commercial DNA-based methods are applied in the analysis of pathogens in routine diagnosis and research. They are growth-independent and, therefore, specifically useful for rapid diagnosis. Moreover, in cases of antibiotic treatment of patients pathogens may persist but not grow in culture. Also, microorganisms may not multiply because growth requirements are not met by the standard media. These non-growing organisms can be detected by DNA-based, molecular methods. Further, microbial communities of medical relevance can be described by powerful approaches using Next Generation Sequencing technologies.

DNA isolation covers an important and critical part of direct molecular analysis of microorganisms in clinical specimens. For this, the method of choice needs to be able to disintegrate a variety of microorganisms from different clades. One way of disrupting microorganisms is mechanical shearing like bead beating. This method is effective, but bears the risk of contamination by handling processes. Also, necessary instrumentation and consumables generate additional costs.

Another approach to a general lysis method is enzymatic degradation of cell walls. Molzym goes this way with a lysis reagent, BugLysis, which is included in their manual and automated microbial enrichment and DNA extraction kits. The BugLysis reagent is being evaluated continuously in studies, employing clinical biopsies, industrial fluids, cell cultures and other specimens. Presently, 1,330 species have been identified by rRNA gene sequencing or specific assay analysis. The species group in one archaeal phylum, 10 bacterial phyla with 302 genera, 39 fungal genera or higher taxonomic levels as well as two protists, Plasmodium falciparum and Plasmodium vivax (download complete list of organisms here). This high diversity of microorganisms demonstrates the broad-range lysing capacity of BugLysis.

The mild lysis procedure provides large DNA fragments (mean size approx. 40-50 kb) that are suitable for certain systems of whole genome sequencing analysis of microbiomes. Importantly, the reagent is under constant quality control ensuring high activity and absence of contaminating microbial DNA.