SpeciesPrimer: A bioinformatics pipeline dedicated to the design of qPCR primers for the quantification of bacterial species
A peer-reviewed article of this Preprint also exists.
Author and article information
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.
Cite this as
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.27870v1note This preprint is not peer-reviewed. You may wish to reference the subsequent peer-reviewed version of this article.
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This is a submission to PeerJ for review.
Sections
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.
Primer sequences of the qPCR assays used for the in vitro validation
Additional Information
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Matthias Dreier conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.
Hélène Berthoud conceived and designed the experiments, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft.
Noam Shani conceived and designed the experiments, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft.
Daniel Wechsler conceived and designed the experiments, authored or reviewed drafts of the paper, approved the final draft.
Pilar Junier conceived and designed the experiments, authored or reviewed drafts of the paper, approved the final draft.
Data Deposition
The following information was supplied regarding data availability:
Code is available at
https://github.com/biologger/speciesprimer
Docker image is available at
Funding
The authors received no funding for this work.
