Comparison of Methods for Isolation of Bacterial and Fungal DNA from Human Blood

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.

Improving molecular diagnosis of neonatal septicaemia: Comparative evaluation of different protocols for extraction of bacterial and fungal DNA from blood samples

“…The use of bead beating was also found useful by some researchers as a method for extraction of pathogenic nucleic acids from microorganisms specially fungi. [5,11] There are many advantages of bead beating like it is faster as well as cheaper than enzymatic treatments, permits safe handling of Samples, allows all the steps to be carried out with a single tube and thus can avoid cross contamination among samples.…”

“…[5] In another study it was 10 3 CFU/ml for gram positive and gram negative bacteria and 10 4 CFU/mL, respectively for fungi. [12] In another study using Taqman based broad range PCR using 16SrRNA gene it was 10 3 CFU/ml for Escherichia coli and Staphylococcus aureus.…”

“…This finding was in concordance to other studies using preliminary blood cell lysis step. [5] We found protocol 2 using whole blood samples, blood cell lysis + step ZR Fungal/Bacterial DNA kit(bead beating+spin column)best in terms of purity and concentration of DNA obtained. The use of bead beating was also found useful by some researchers as a method for extraction of pathogenic nucleic acids from microorganisms specially fungi.…”

“…Gram-positive bacteria, Gramnegative bacteria, yeast or filamentous fungi. [4,5,6] In scientific literature, methods for bacterial and fungal DNA isolation from blood are described, but many are enabling one to separately isolate either bacterial or fungal DNA from blood. [4,7] It is necessary to compile several processes, for obtaining high-quality DNA matrix.…”

A novel, nested, multiplex, real-time PCR for detection of bacteria and fungi in blood

“…DNA isolation was carried out according to the method described by Gosiewski et al with the employment of a ready-to-use Blood Mini (A&A Biotechnology) kit [4]. The same method was used to isolate DNA from blood samples of patients with clinical symptoms of sepsis.…”