ProbioticChip: A novel DNA microarray for accurate and efficient subspecies-level identification of probiotics

The effects and efficacy of probiotics on the host depend on precise bacterial composition, making accurate taxonomic identification important for product quality control. Conventional approaches, including polyphasic methods (phenotype and chemotaxonomy) and 16S rRNA gene sequencing, are often limited in resolution, throughput, or quantitative accuracy. To address these limitations, we developed ProbioticChip, a DNA microarray comprising 13,318 probes targeting 37 commonly used probiotic taxa at the species and subspecies levels. Validation using 16 mock probiotic communities comprising 67 strains demonstrated that ProbioticChip can sensitively detect target taxa with high specificity. Quantitative evaluation further revealed a strong linear relationship between total probe fluorescence intensity and DNA input (R 2 > 0.90 across the range of 0.05–10 ng), indicating its sensitivity and potential utility for quantitative analysis. We further applied ProbioticChip to five commercial probiotic products, achieving species- and subspecies-level identification with preliminary quantitative estimates within three days. Together, these results indicate that ProbioticChip represents a rapid and high-throughput platform for compositional analysis of probiotic products, and may support applications in quality assessment and regulatory monitoring. • ProbioticChip enables rapid subspecies-level identification of probiotics. • 13,318 probes designed for 37 probiotic taxa with high specificity and accuracy. • Quantitative detection achieved with strong linear correlation to DNA input. • Outperforms 16S sequencing in resolving closely related probiotic subspecies. • Provides a practical tool for probiotic quality control and authenticity testing.