SpeciesPrimer: A bioinformatics pipeline dedicated to the design of qPCR primers for the quantification of bacterial species
- Published
- Accepted
- Subject Areas
- Bioinformatics, Food Science and Technology, Microbiology
- Keywords
- Primer design, Species specific quantification, Quantitative Real-Time Polymerase Chain Reaction, qPCR primer, Species specific sequences, Docker container
- Copyright
- © 2019 Dreier et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2019. SpeciesPrimer: A bioinformatics pipeline dedicated to the design of qPCR primers for the quantification of bacterial species. PeerJ Preprints 7:e27870v1 https://doi.org/10.7287/peerj.preprints.27870v1
Abstract
Background. 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 reactants. In order to develop a high-throughput qPCR approach for the rapid and cost-efficient quantification of microbial species in a given system (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.
Results. 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 Enterococcus faecium, Enterococcus faecalis, Pediococcus acidilactici and Pediococcus pentosaceus. Selected primers were first evaluated in silico and subsequently in vitro 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.
Conclusion. 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.
Author Comment
This is a submission to PeerJ for review.
Supplemental Information
Pipeline input (Input genome assembly accessions, Pipeline configuration, Specieslist)
Bacterial strains (Target strains, Non-target strains, Cultivation conditions)
qPCR raw data
Raw data of qPCR experiments. Data export from Rotor-Gene 6000 Software 1.7 with dynamic tube normalization and a threshold of 0.05 for quantification cycle (Cq) value calculation, the five first cycles were ignored for the determination of the Cq values. The peak calling threshold for the melt curve analysis was set to -2 dF/dT and a temperature threshold was set 2 °C lower than the positive control peak.