Melting-Curve qPCR-based to detect Enterococcus and Veillonella genus in human gut microbiota
DOI:
https://doi.org/10.11606/issn.1679-9836.v104iesp.e-242581Keywords:
Gastrointestinal Microbiome, Real-time Polymerase Chain Reaction, Enterococcus, Veillonella, Nucleic Acid DenaturationAbstract
The human gut microbiota is composed of about 100 trillion organisms, such as bacteria, viruses, protozoa, and fungi, that live in symbiosis with the host. These microorganisms play an essential role in maintaining intestinal homeostasis and preventing diseases. However, changes in its composition lead to a state of imbalance, dysbiosis, which is associated with inflammatory bowel diseases and colorectal cancer. Among the genera of interest, Enterococcus spp. and Veillonella spp. have distinct functions related to intestinal pH regulation, nutrient synthesis, and lactate fermentation. The study aimed to develop a method based on real-time PCR (qPCR) for the qualitative detection of these two bacterial genera in the human gut microbiota. For this, 13 and 27 ribosomal RNA gene sequences of Enterococcus and Veillonella, respectively, were retrieved from the GenBank database, used to create a nconsensus sequence and design specific primer pairs. The reactions were conducted in a Rotor-Gene Q-5Plex HRM thermocycler, with an optimized girdling temperature of 58 °C and 56 °C, and peak dissociation determined at 82,1 °C and 82 ºC for Enterococcus and Veillonella, respectively. The methodology demonstrated efficacy and specificity in the identification of microorganisms, validating the use of qPCR as a viable and lower-cost alternative to sequencing techniques for taxonomic analysis of intestinal bacteria.
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Copyright (c) 2025 Thiago França-Soares, Bruno Shimokava, Alessandra Sonomura, Artur N Mizuno, Heloísa Borges da Silveira, Lucy Megumi Yamauchi, Sueli Fumie Yamada-Ogatta, Eliandro Tavares
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