Fig. 2: Probe design and validation.

A Schematic of the MicroCart in silico probe design process. A curated database of 16S rRNA sequences from intestinal microbiota was first created, then probe candidates were designed that are specific for target bacterial groups. Stringent criteria, including melting temperature, hybridization efficiency, secondary structure, coverage, and specificity were used to select for top candidate probes. Optionally, the MicroCart probe design tool can also create combination sets of probes to maximize performance. Created in BioRender. Zhu, B. (2024) https://BioRender.com/h69v030. B An illustration of the experimental validation process for designed probes. Bacteria strains were cultured, harvested, fixed (using our optimized MFPE fixation), and embedded in HistoGel. Subsequently, HistoGel-bacteria strains were dehydrated and embedded in paraffin in a microarray fashion, into Bacteria MicroArrays (BMA), before sectioning onto slides. Probe validation can be either performed using primary oligos conjugated to fluorophores, or using a secondary barcoded-oligos conjugated to fluorescence to reduce cost through flexibility and increase efficiency through multiplexing. C Left: Experimental validation on BMA slides, with probes designed to target phylum groups (Bacteroidetes or Firmicutes). Right: probes designed to target various probiotic species. Experiments were repeated three times to insure robustness.