A rapid method for species-level identification of Bacillus subtilis group strains via multiplex pan-genome-based PCR

The various species within the Bacillus subtilis group possess significant economic and practical value, but exhibit distinct functions and have divergent applications. Thus, there is an urgent need for a rapid, efficient, and cost-effective method to accurately distinguish species and their strains within the B. subtilis group, to enable bacterial resources to be precisely screened and classified. Here, we developed a comparative genomics-based multiplex PCR method for the species-level identification of B. subtilis group strains to facilitate the accumulation of B. subtilis group microbial resources. We constructed a pan-genome from 753 genomes spanning 12 species within the B. subtilis group, and used it to identify six marker genes that together could distinguish among the species: araE, ykqA, yicL, corC, gtaB, and gdpP. Specific primers for these marker genes were designed to generate distinct PCR product profiles that enabled species-level identification of strains. This method was used to successfully identify 21 B. subtilis group strains from a collection of 23 bacterial isolates obtained from soil, yielding an accuracy rate of 91.30%. Our results demonstrate the feasibility and reliability of this multiplex PCR approach, which could provide an efficient and accurate tool for screening and classifying B. subtilis group strains from soil microbial communities, and thereby support their exploration and utilization in various applications.