A Validated Method for Detection of <i>Bifidobacterium infantis</i> Bi-26 ^TM DNA in Stool by Quantitative Real-Time PCR

Abstract Background Bifidobacterium longum subspecies infantis ( B. infantis ) is a commensal bacterium enriched in the microbiota of breastfed infants and associated with beneficial effects on infant gut health. Clinical studies evaluating B. infantis supplementation in infants have reported increased colonization, reduced inflammation, enhanced vaccine responsiveness, and improved growth outcomes. To support rigorous evaluation of these effects in further clinical trials, reliable and validated methods for quantifying B. infantis in stool are needed. Methods We validated a quantitative real-time PCR (qPCR) assay against a conserved B. infantis genomic target. Assay performance was evaluated using DNA from the B. infantis strain Bi-26 TM , spiked infant stool pools, and matrix controls. Validation parameters included linearity, sensitivity, accuracy, within- and between-run reproducibility, recovery, and matrix interference, with acceptance criteria informed by bioanalytical best practices. Results The assay demonstrated robust performance across validation parameters. Linearity was confirmed over at least six orders of magnitude (R 2 &gt; 0.99; mean efficiency 96.4%), with a lower limit of quantification of 37.36 genome equivalents / reaction. Analytical sensitivity was high, with a 95% limit of detection of 4.67 genome equivalents / reaction. Accuracy across multiple stool pools met acceptance criteria, with mean deviation from nominal values of 0.39 log 10 . Reproducibility was strong, with within-run variability of 0.37% and between-run variability of 1.78%. Recovery averaged 41.3%, exceeding the predefined minimum threshold, and matrix interference was minimal. Conclusions This study reports a validated qPCR assay suitable for quantifying B. infantis Bi-26 DNA in stool samples collected in stabilized formats. Its robust analytical performance supports its use in clinical and translational research evaluating B. infantis colonization and its health impacts in early infant life.