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

Abstract

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.