Bifidobacteria are the representatives of the phylum Actinomycetota in the microbiocenosis of the human large intestine, a characteristic of its condition which play the role of a functional dominant in it. Microbiome studies of the human intestine, carried out with the use of molecular genetic methods, have demonstrated the distribution of the number of identified species of the phylum Actinomycetota with the results of the bacteriological method.
THE AIM OF THE STUDY
Was the determination of the effectiveness of some methods of sample preparation of faeces for the isolation of DNA of bifidobacteria, cutibacteria and bacteroids.
MATERIALS AND METHODS
The study was carried out on faecal samples obtained from apparently healthy adult subjects treated with various suspension media (isotonic sodium chloride solution or sodium phosphate buffer), fractionated by centrifugation and subjected to total DNA extraction from two different fractions: unstained superficial and deep fibrous. The concentration of isolated DNA was determined by the spectrophotometric method. The isolated DNA was used as a DNA template during PCR analysis in the developed test system for the generic identification of bifidobacteria, cutibacteria, and bacteroids.
RESULTS AND DISCUSSION
The use of phosphate buffer or simply isotonic sodium chloride solution does not significantly affect the efficiency of DNA extraction. PCR analysis using genus-specific primers for the small ribosomal RNA gene and using a similar bacteroid detection system as a control shows that the use of phosphate buffer or isotonic sodium chloride solution at the stage of resuspension of the test material does not affect the course of the amplification reaction. At the same time, DNA isolation from the upper sediment fraction, as recommended by the kit manufacturer, does not allow detecting bifidobacteria in it, while amplification of bacteroid DNA is successful in all cases. Bifidobacteria DNA is detected only in the deep fibrous phase of feces.
CONCLUSIONS
The data obtained demonstrate the importance of taking into account the ability of such indigenous human intestinal microorganisms as bifidobacteria to concentrate mainly in the fibrous part of feces, which is not always a source of bacterial DNA isolation.
