Abstract
BackgroundQuantitative 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.ResultsWe 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.ConclusionIn 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.
Highlights
Quantitative real-time PCR is a well-established method for the detection and quantification of bacteria in microbiology, for instance in the context of pathogen detection in clinical and veterinary diagnostics and food safety (Cremonesi et al 2014; Curran et al 2007; Garrido-Maestu et al 2018; Ramirez et al 2009)
The species list consisted of 259 species and subspecies names detected in dairy products, namely from species names collected from data of 16S rRNA meta-genome sequencing studies in milk and cheese varieties (Marco Meola Agroscope, pers. comm.) and dairy-related bacteria from the list of bacterial species and subspecies with technological beneficial use in food products (Almeida et al 2014)
Primer design for four bacterial species commonly found in cheese was performed with the SpeciesPrimer pipeline
Summary
Quantitative real-time PCR (qPCR) is a well-established method for the detection and quantification of bacteria in microbiology, for instance in the context of pathogen detection in clinical and veterinary diagnostics and food safety (Cremonesi et al 2014; Curran et al 2007; Garrido-Maestu et al 2018; Ramirez et al 2009). 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. Most available tools for primer design require either laborious manual manipulation or high-performance computing systems
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