Seleção de primers para detecção de bactérias probióticas por meio do método de qPCR

Abstract

The importance of the intestinal microbiota for human health, understanding its population dynamics, its interaction with the host, and its influence on the functioning of the human organism have been the focus of intense research over the last 10 years. The activity of intestinal microorganisms is also relevant in modulating mucosal immunity, reducing markers of chronic inflammation, and facilitating the competitive exclusion of pathogens. Thus, modulating the intestinal microbiota has been considered a strategy for promoting health and helping to reduce the risk of diseases. Regularly consuming probiotic and prebiotic foods alters the intestinal microbiota's composition and metabolic activity. Researchers have developed several models and methodologies for in vitro simulation of digestion under the conditions of the human gastrointestinal tract to elucidate how passage through the gastrointestinal tract affects foods and food constituents, probiotic microorganisms, and drugs, which may support the development of functional foods. In this context, fructooligosaccharides (FOS), which are widespread carbohydrates that promote the health of the intestinal microbiota by increasing the levels of Bifidobacterium and Lactobacillus, were used as a prebiotic supplement to improve the health of the intestinal flora. In order to detect and quantify the increase in the levels of these probiotics, primers already described in the literature were tested, in addition to new ones designed to improve specificity, since the primer design step is essential for the technique Quantitative Polymerase Chain Reaction (qPCR) and characteristics such as primer length, melting temperature (Tm), annealing temperature, amplicon size and percentage of GC content determine the specificity of the primer, which in turn determines the accuracy and reproducibility of the results. Low-specificity primers can bind to non-target sequences, amplifying nonspecific products that can impair quantitative data analysis. On the other hand, specific primers amplify only the desired sequence, ensuring accurate results. The qPCR requires specific amplification of the sequence of interest to produce reliable results. The previous study of primer efficiency allowed the quantification and comparison of probiotic levels before and after the use of FOS in the in vitro assay.