Generic workflow for a rapid and easy design of strain-specific PCR and qPCR primers, applied to the assessment of bacterial strains survival in soil

Background Detecting bacteria at the strain level is crucial in microbiology. Although qPCR is widely used, designing strain-specific primers remains a challenge due to nucleotide sequence similarities among related strains. Methods and Results This paper introduces a simplified, web-based workflow for designing strain-specific primers using publicly available microbial genomes. The method does not require advanced bioinformatics skills and can be applied using a basic computer. Primers designed using this workflow are applied to assess the survival of two close Bacillus strains in soil microcosms. Conclusion The workflow offers an accessible solution for accurate bacterial strain detection, and fills a gap for researchers without specialized training in bioinformatics.