Abstract
The study aimed at optimization of DNA isolation from blood of representatives of four microbial groups causing sepsis, i.e., Gram negative: Escherichia coli, Gram positive: Staphylococcus aureus, yeast: Candida albicans, and filamentous fungus: Aspergillus fumigatus. Additionally, the five commercial kits for microbial DNA isolation from the blood were tested. The developed procedure of DNA isolation consisted of three consecutive steps, i.e., mechanical disruption, chemical lysis, and thermal lysis. Afterward, DNA was isolated from the previously prepared samples (erythrocyte lysis) with the use of five commercial kits for DNA isolation. They were compared paying heed to detection limit, concentration, DNA purity, and heme concentration in samples. The isolation of DNA without preliminary erythrocyte lysis resulted in far higher heme concentration than when lysis was applied. In the variant with erythrocyte lysis, two of the commercial kits were most effective in purifying the DNA extract from heme. Designed procedure allowed obtaining microbial DNA from all four groups of pathogens under study in the amount sufficient to conduct the rtPCR reaction, which aimed at detecting them in the blood.
Highlights
Effective diagnostic of factors causing the infection is the most important, and most difficult, problem in the treatment of blood infections
The study aimed at optimization of DNA isolation from blood of representatives of four microbial groups causing sepsis, i.e., Gram negative: Escherichia coli, Gram positive: Staphylococcus aureus, yeast: Candida albicans, and filamentous fungus: Aspergillus fumigatus
Four strains were used in the research, these were representing groups diversified as regards the cell wall structure: Gram-negative Escherichia coli ATCC 25922 (American Type Culture Collection), Gram-positive Staphylococcus aureus ATCC 33497, Candida albicans ATCC 10231 yeast, and Aspergillus fumigatus ATCC 14110 filamentous fungus
Summary
Effective diagnostic of factors causing the infection is the most important, and most difficult, problem in the treatment of blood infections. It is decisive as regards the effectiveness of therapy and, the costs and the duration of hospitalization. The so-called diagnostic ‘‘gold standard’’ has been constituted by blood cultures carried out on special growth media, preferably in automated cell culture systems. The advantages of such methods are their simplicity and relatively low costs of testing. Their weakness is that they are time-consuming, taking up to 5 days (until the test results are issued), and have low detection limit, which causes only 15–20 % of the culture to obtain microbial growth [3]
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