Standardizing oral microbiome sampling for qPCR: methodological and exploratory insights into nutritional status

Abstract Standardization of oral sample collection methods is essential for accurate and reproducible microbiota quantification. This methodological study aimed to evaluate different oral collection methods to identify the most consistent approach for bacterial quantification by qPCR using samples from adolescent individuals. In addition, to assess the biological applicability of the best method, an exploratory analysis compared bacterial profiles between eutrophic and overweight/obese adolescents and explored associations between bacterial abundance and body composition parameters. Samples of unstimulated saliva, cheek swabs, and biofilm were collected from the same individuals, and qPCR was used to quantify total bacteria (16 S rRNA gene), Bacillota , and Bacteroidota phyla. Unstimulated saliva produced the lowest variability in bacterial quantification compared with other methods ( p < 0.05). Moderate correlations were observed between saliva and biofilm, whereas saliva and cheek swab showed weak associations. Although bacterial copy numbers tended to be higher in overweight/obese individuals, these differences were not statistically significant. Correlation matrices suggested group-specific associations between bacterial taxa and body composition parameters, demonstrating the potential of saliva for microbiome assessment in studies of nutritional and metabolic health. This study validated unstimulated saliva as a reproducible, non-invasive, and cost-effective biofluid for oral microbiota quantification by qPCR. The method provides consistent results suitable for large-scale, translational, or point-of-care applications.