Design of Bacterial Strain-Specific qPCR Assays Using NGS Data and Publicly Available Resources and Its Application to Track Biocontrol Strains

Biological control is emerging as a feasible alternative to chemical pesticides in agriculture. Measuring the microbial biocontrol agent (mBCA) populations in the environment is essential for an accurate environmental and health risk assessment and for optimizing the usage of an mBCA-based plant protection product. We hereby show a workflow to obtain a large number of qPCR markers suitable for robust strain-specific quantification. The workflow starts from whole genome sequencing data and consists of four stages: (i) identifying the strain-specific sequences, (ii) designing specific primer/probe sets for qPCR, and (iii) empirically verifying the performance of the assays. The first two stages involve exclusively computer work, but they are intended for researchers with little or no bioinformatic background: Only a knowledge of the BLAST suite tools and work with spreadsheets are required; a familiarity with the Galaxy environment and next-generation sequencing concepts are strongly advised. All bioinformatic work can be implemented using publicly available resources and a regular desktop computer (no matter the operating system) connected to the Internet. The workflow was tested with five bacterial strains from four different genera under development as mBCAs and yielded thousands of candidate markers and a triplex qPCR assay for each candidate mBCA. The qPCR assays were successfully tested in soils of different natures, water from different sources, and with samples from different plant tissues. The mBCA detection limits and population dynamics in the different matrices are similar to those in qPCR assays designed by other means. In summary, a new accessible, cost-effective, and robust workflow to obtain a large number of strain-specific qPCR markers is presented.