Selection and Optimization of High-Yielding DNA Isolation Protocol for Quantitative Analyses of Methanogenic Archaea.

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.