Single-cell rapid identification, in situ viability and vitality profiling, and genome-based source-tracking for...

Rapid expansion of the probiotics industry demands fast, sensitive, comprehensive, and low-cost strategies for quality assessment. Here, we introduce a culture-free, one-cell-resolution, phenome-genome-combined strategy called Single-Cell Identification, Viability and Vitality tests, and Source-tracking (SCIVVS). For each cell directly extracted from the product, the fingerprint region of D₂O-probed single-cell Raman spectrum (SCRS) enables species-level identification with 93% accuracy, based on a reference SCRS database from 21 statutory probiotic species, whereas the C-D band accurately quantifies viability, metabolic vitality plus their intercellular heterogeneity. For source-tracking, single-cell Raman-activated Cell Sorting and Sequencing can proceed, producing indexed, precisely one-cell-based genome assemblies that can reach ~99.40% genome-wide coverage. Finally, we validated an integrated SCIVVS workflow with automated SCRS acquisition where the whole process except sequencing takes just 5 h. As it is >20-fold faster, >10-time cheaper, vitality-revealing, heterogeneity-resolving, and automation-prone, SCIVVS is a new technological and data framework for quality assessment of live-cell products.