Comparative Evaluation of DNA Extraction Methods from Fecal Samples: Statistical Analysis of Commercial Kits and Laboratory Protocols Using Real-Time PCR Data

Emergence of new data on the association between composition of the intestinal microbiota and various human diseases have generated increasing interest in its investigation. In this context, selection of the DNA extraction method represents a critical stage in experimental design, significantly affecting the reliability and reproducibility of results. This study presents a comparative analysis of 12 DNA extraction methods applied to 46 fecal samples, including 9 commercial kits and 3 laboratory protocols. We evaluated taxonomic representation, including Gram-positive (Lactobacillaceae, Coprococcus spp., Streptococcus sp., Clostridium leptum) and Gram-negative bacteria (Enterobacteriaceae, Akkermansia muciniphila, Fusobacterium nucleatum, Bacteroides fragilis). Extraction efficiency was assessed by DNA yield, expressed in GE/μL of eluate or in GE/g of feces, as well as by the frequency of low-abundance taxa loss. Based on the lysis type, clustering of methods was demonstrated: mechanical lysis provided stable and high DNA yields, particularly for Gram-positive bacteria, while chemical and enzymatic methods showed lower efficiency. We determined that lysis type and preliminary whole-fecal sample preparation are the key factors affecting DNA extraction efficiency and preservation of the native taxonomic profile. The best results were demonstrated by the QIAamp® PowerFecal® Pro DNA Kit (Qiagen) and the combination of AmpliTest UniProb + AmpliTest RIBO-prep kits (Centre for Strategic Planning, of the Federal medical and biological agency, Russia), both of which outperformed other methods in terms of DNA yield. The QIAamp® Fast DNA Stool Mini Kit (Qiagen) showed minimal losses of low-abundance taxa. These findings can be used for the standardization of extraction methodologies and the development of domestic protocols.