Stool samples are widely used as a non-invasive source for molecular biology research, containing microbial DNA, food-derived DNA and host-derived DNA. However, stool DNA extraction is challenged by low host DNA content, partial nucleic acid degradation and, most critically, the presence of a wide range of PCR inhibitors.

Stool samples typically contain polysaccharides, bile salts, bile pigments and other digestion-derived compounds that can interfere with enzymatic reactions and reduce downstream detection efficiency. Effective DNA extraction therefore requires not only efficient cell disruption, but also reliable removal of inhibitory substances.

The BioString^TM Stool DNA Kit is developed to address these challenges through a workflow combining bead-assisted mechanical lysis with adsorbent-based purification. The system is designed to reduce inhibitor background while maintaining DNA recovery from complex fecal matrices.

BioString^TM Stool DNA Kit performance was evaluated using stool samples from multiple species, including healthy human stool samples, bovine stool samples, chicken stool samples and dog stool samples.

Nanodrop analysis showed that purified stool DNA reached suitable purity for downstream molecular workflows across different sample matrices. DNA recovery varied among sample types, reflecting expected differences in microbial content, diet, host cell contribution and sample storage condition. This is consistent with the biological variability normally observed in stool DNA extraction.

Genomic DNA integrity was assessed by loading 10 μL purified DNA on a 0.8% agarose gel. The electrophoresis results showed recovery of relatively intact genomic DNA from the tested stool samples. Lower recovery observed in some chicken stool samples was attributed in the report to longer sample storage, indicating that stool preservation remains an important pre-analytical factor for reliable DNA recovery.

PCR compatibility was verified using 5 μL purified stool DNA as template for bacterial 16S amplification. A 200 bp bacterial target fragment was successfully amplified from human, bovine, chicken and dog stool DNA, confirming that the purified DNA could be used directly for bacterial DNA detection without additional cleanup.

Inhibitor removal was further assessed using a low-input spike-in PCR test. In this assay, 20 ng human genomic DNA was added to 5 μL purified stool DNA and used as template for amplification of a 200 bp human target fragment. Clear amplification was obtained in the first PCR round, indicating effective reduction of stool-derived PCR inhibitors and supporting the use of purified DNA in sensitive PCR-based workflows.

Proteinase K and Buffer PCI should be stored at 2-8°C upon arrival. However, short-term storage (up to 12 weeks) at room temperature (15-25°C) does not affect their performance. The remaining kit components can be stored at room temperature (15-25°C) and are stable for at least 18 months under these conditions. The entire kit can be stored at 2–8°C, but in this case buffers should be redissolved before use. Make sure that all buffers are at room temperature when used.