Pangenome-based design of strain-specific primers enables precise monitoring of bacteria in human microbiome intervention trials.

Lactic acid bacteria (LAB), which include many well-known beneficial bacteria, are widely used in food fermentation, biotechnology, and probiotic formulations. Within this group, strains of Lactobacillaceae are particularly well studied. However, accurate monitoring of the survival and persistence of individual strains across different niches remains challenging due to the limited availability of strain-specific detection tools. This study introduces a robust pangenome-based approach to identify unique genetic targets for the development of strain-specific primers. Using this approach, selective and specific primers were developed for six strains spanning different LAB species. Primers for the widely used probiotic strains Lacticaseibacillus rhamnosus GG and Lactiplantibacillus plantarum WCFS1 were validated using samples from three placebo-controlled human intervention studies. These assays enabled precise tracking of each strain across body sites, including the skin and the upper respiratory tract, revealing clear distinctions between treatment and control samples. This gene-targeted qPCR method provides a sensitive and reliable method for strain-level detection and can be readily adapted to other bacterial strains for diverse research and industrial applications.