Kiefer, A. | Search bioRxiv

bioRxiv2025

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

BackgroundBifidobacterium 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. MethodsWe 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-26TM, 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. ResultsThe assay demonstrated robust performance across validation parameters. Linearity was confirmed over at least six orders of magnitude (R2 > 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 log10. 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. ConclusionsThis 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.

microbiology↗

bioRxiv2021

Production and secretion of functional full-length SARS-CoV-2 spike protein in Chlamydomonas reinhardtii

The spike protein is the major protein on the surface of coronaviruses. It is therefore the prominent target of neutralizing antibodies and consequently the antigen of all currently admitted vaccines against SARS-CoV-2. Since it is a 1273-amino acids glycoprotein with 22 N-linked glycans, the production of functional, full-length spike protein was limited to mammalian and insect cells, requiring complex culture media. Here we report the production of full-length SARS-CoV-2 spike protein - lacking the C-terminal membrane anchor - as a secreted protein in the prefusion-stabilized conformation in the unicellular green alga Chlamydomonas reinhardtii. We show that the spike protein is efficiently cleaved at the furin cleavage site during synthesis in the alga and that cleavage is abolished upon mutation of the multi-basic cleavage site. We could enrich the spike protein from culture medium by ammonium sulfate precipitation and demonstrate its functionality based on its interaction with recombinant ACE2 and ACE2 expressed on human 293T cells. Chlamydomonas reinhardtii is a GRAS organism that can be cultivated at low cost in simple media at a large scale, making it an attractive production platform for recombinant spike protein and other biopharmaceuticals in low-income countries.

synthetic biology↗