Abstract
Methanogenic archaea are a functionally important component of the intestinal microbiota of humans and animals, participating in the utilization of detrimental hydrogen produced during gut fermentation. Despite this, archaeal DNA has rarely been found in intestinal microbiome analyses, which prompts the need to optimize detecting procedures of these microorganisms, including the DNA isolation step. Three commercially available kits for DNA isolation and one extra purification kit that removes PCR inhibitors were evaluated on chicken droppings. In addition, different variants of mechanical lysis and a double elution were tested to ensure the maximum efficiency of DNA isolation from archaea as well as bacteria. A quantitative real-time PCR was used to monitor the optimization progress. As a result, the combination of the selected Genomic Mini AX Bacteria+ kit with a 2-min-long sonication by ultrasonic probe and enzymatic pretreatment gave excellent extraction efficiency rates for DNA of methanogenic archaea (an approximate 50-fold increase compared to the standard enzymatic lysis described by the producer) and, at the same time, provided optimal protection of DNA extracted from bacteria susceptible to enzymatic lysis. The presented results indicate that the optimized protocol allows for highly efficient extraction of total DNA, which is well-suited for quantitative microbial analyses by real-time PCR.
Highlights
That DNA isolation protocols should be designed to include as many microorganisms as possible; bacteria but archaea and single-celled eukaryotes as well
Eight out of 16 samples after both single and double purification were negative for methanogenic archaea
The purpose of this study was to assess the efficiency of purification of DNA isolated was used for further experiments and inoculated with a known amount of mcrA-positive using selected kits, as well as the real-time PCR performance in detecting of methanogens plasmid
Summary
The gut microbiome has recently been gaining more and more attention due to the increasing awareness of the microbiota’s role in maintaining the host’s health and wellbeing [1]. A difference between health and illness often relies on a quantitative microbial imbalance For this reason, quantitative studies of microbiota require proper isolation of total DNA from stool or intestinal samples. Quantitative studies of microbiota require proper isolation of total DNA from stool or intestinal samples It stands to reason, that DNA isolation protocols should be designed to include as many microorganisms as possible; bacteria but archaea and single-celled eukaryotes as well. That DNA isolation protocols should be designed to include as many microorganisms as possible; bacteria but archaea and single-celled eukaryotes as well Out of these three, archaea seem to be the most difficult to extract DNA from. Murein undergoes degradation by lysozyme at the 1,4glycosidic bond site between N-acetylglucosamine (NAG) and N-acetylmuramic (NAM)
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