SpeciesPrimer: a bioinformatics pipeline dedicated to the design of qPCR primers for the quantification of bacterial species

• Published in: PeerJ (San Francisco, CA) Vol. 8; p. e8544
• Main Authors: Dreier, Matthias, Berthoud, Hélène, Shani, Noam, Wechsler, Daniel, Junier, Pilar
• Format: Journal Article
• Language: English
• Published: United States PeerJ. Ltd 18.02.2020; PeerJ, Inc; PeerJ Inc
• Subjects: Automation; Bacteria; Batch processing; Bioinformatics; Cheese; Computational biology; Dairy products; Design; DNA; Docker container; Fermented food; Food microbiology; Food Science and Technology; Genes; Genomes; High-throughput screening; Identification; Microbiology; Microfluidics; Pathogens; Pipelines; Polymerase chain reaction; Primer design; Primers; qPCR primer; Quality control; Quantitative real-time polymerase chain reaction; Reagents; Species; Species specific quantification; Species specific sequences; Taxonomy; Time; Workflow software; Species specific quantification; Bioinformatics pipeline; Primer validation; Species specific sequences; Quantitative real-time polymerase chain reaction; Docker container; qPCR primer; Primer design
• ISSN: 2167-8359; 2167-8359
• DOI: 10.7717/peerj.8544

Quantitative real-time PCR (qPCR) is a well-established method for detecting and quantifying bacteria, and it is progressively replacing culture-based diagnostic methods in food microbiology. High-throughput qPCR using microfluidics brings further advantages by providing faster results, decreasing the costs per sample and reducing errors due to automatic distribution of samples and reagents. In order to develop a high-throughput qPCR approach for the rapid and cost-efficient quantification of microbial species in complex systems such as fermented foods (for instance, cheese), the preliminary setup of qPCR assays working efficiently under identical PCR conditions is required. Identification of target-specific nucleotide sequences and design of specific primers are the most challenging steps in this process. To date, most available tools for primer design require either laborious manual manipulation or high-performance computing systems. We developed the SpeciesPrimer pipeline for automated high-throughput screening of species-specific target regions and the design of dedicated primers. Using SpeciesPrimer, specific primers were designed for four bacterial species of importance in cheese quality control, namely , , and . Selected primers were first evaluated and subsequently using DNA from pure cultures of a variety of strains found in dairy products. Specific qPCR assays were developed and validated, satisfying the criteria of inclusivity, exclusivity and amplification efficiencies. In this work, we present the SpeciesPrimer pipeline, a tool to design species-specific primers for the detection and quantification of bacterial species. We use SpeciesPrimer to design qPCR assays for four bacterial species and describe a workflow to evaluate the designed primers. SpeciesPrimer facilitates efficient primer design for species-specific quantification, paving the way for a fast and accurate quantitative investigation of microbial communities.