Novel primers to identify a wider diversity of butyrate-producing bacteria.

Butyrate-producing bacteria are a functionally important part of the intestinal tract flora, and the resulting butyric acid is essential for maintaining host intestinal health, regulating the immune system, and influencing energy metabolism. However, butyrate-producing bacteria have not been defined as a coherent phylogenetic group. They are primarily identified using primers for key genes in the butyrate-producing pathway, and their use has been limited to the Bacillota and Bacteroidetes phyla. To overcome this limitation, we developed functional gene primers able to identify butyrate-producing bacteria through the butyrate kinase gene, which encodes the enzyme involved in the final step of the butyrate-producing pathway. Genomes extracted from human and rat feces were used to amplify the target genes through PCR. The obtained sequences were analyzed using BLASTX to construct a developmental tree using the MEGA software. The newly designed butyrate kinase gene primers allowed to recognize a wider diversity of butyrate-producing bacteria than that recognized using currently available primers. Specifically, butyrate-producing bacteria from the Synergistota and Spirochaetota phyla were identified for the first time using these primers. Thus, the developed primers provide a more accurate method for researchers and doctors to identify potential butyrate-producing bacteria and deepen our understanding of butyrate-producing bacterial species.