Researchers from the Molecular Biology Unit of the R&D Department of Futureco Bioscience have published their most recent success in the scientific journal Frontiers in Microbiology. The article “Design of specific qPCR assays for bacterial strains using state-of-the-art sequencing data and publicly available resources, and their application for monitoring bacterial strains for biocontrol”, can be consulted here. It describes and explains the workflow, based on next-generation sequencing (#NGS), to design robust markers for the detection and quantification of specific bacterial strains. The protocol is designed to minimize the laboratory workload and to use bioinformatic tools that are accessible to all researchers.
The workflow starts with the whole genome NGS sequencing data and consists of three stages: i) Identification of strain-specific sequences, ii) Design of specific primer/probe sets for the qPCR, and iii) Empirical verification of the assay performance. The first two stages involve exclusively computer work with little or no bioinformatics training since the only specific prerequisites are knowledge of the BLAST family tools and the ability to work with spreadsheets. All bioinformatic work can be carried out using publicly available resources and a common desktop computer (regardless of the operating system) connected to the Internet. The described workflow has been verified in the laboratory with five bacterial strains of four different genera under development in our facilities as microbial biocontrol agents. The qPCR assays have been successfully carried out in soil, water and plant tissues of different natures. The detection limits and population dynamics in the different matrices are comparable to assays designed by other means. In summary, our scientists have designed an accessible, cost-effective and robust new workflow to obtain a large number of strain-specific robust markers for qPCR assays.
