![]() We aimed to design a series of primers that function with the same qPCR cycling conditions and primer concentrations for later usage in a high-throughput microfluidic qPCR platform. It identifies sequences present in all input genomes and designs specific primers accordingly.įdp find_differential_primers x Feature supported – Feature not supported * Access has to be requested QC Quality control CDS Coding sequences TOPSI is an automated high-throughput pipeline for the design of primers, primarily developed for pathogen-diagnostic assays. ![]() Similarly, the fdp pipeline designs primers for a set of positive genomes and, further, allows to extract primers specific to subclasses of the positive set and performs specificity check against a negative set of genomes. It designs primers for the core sequences and validates them with an in silico PCR validation method against the positive and negative reference sets. RUCS is able to identify unique core sequences in a positive set of genomes (target) compared to a negative set of genomes (non-target). Tools and pipelines that encompass both the identification of target sequences from bacterial draft genomes and the design of primer candidates include, for instance, RUCS, the find_differential_primers (fdp) pipeline and TOPSI ( Pritchard et al., 2012 Thomsen et al., 2017 Vijaya Satya et al., 2010). PrimerServer ( Zhu et al., 2017) allows to design primers for multiple sites across a whole genome sequence and performs a specificity check. PrimerMiner ( Elbrecht, Leese & Bunce, 2017) is a tool that automatically downloads sequences of marker genes for taxonomic groups specified by the user and creates alignments and consensus sequences as target sequences for the design of degenerate primers. ![]() Table 1 provides an overview of the features of different primer design tools and pipelines. Primer3 predicts suitable PCR primers for an input target sequence, while Primer-BLAST combines Primer3 with a BLAST search in a selected nucleotide sequence database to assess the specificity of the primers for the target sequence. Various commercial and open source programs facilitate the design of specific primers for a target sequence, such as the standard tools Primer3 and Primer-BLAST ( Untergasser et al., 2012 Ye et al., 2012). ![]() This, in combination with the increased computing power, makes it now possible to screen and compare hundreds of genomes and to predict unique target sequences in a relatively short time. Today, the steadily increasing number of prokaryotic draft genomes facilitates the identification of new and unique target regions. Further, housekeeping genes such as, for instance, tuf, recA and pheS, were successfully used as target sequences for a variety of bacterial species in fermented foods ( Falentin et al., 2010 Masco et al., 2007 Scheirlinck et al., 2009). However, the regions that are targeted in the 16S rRNA gene often do not provide sufficient resolution to differentiate between closely related bacterial species ( Moyaert et al., 2008 Torriani, Felis & Dellaglio, 2001 Wang et al., 2007). Before microbial draft genomes became widely available, the 16S rRNA gene was frequently used as a target sequence. The main challenges for the successful development of any qPCR assay are the identification of a specific target nucleotide sequence and the design of primers that bind exclusively to that target sequence. Therefore, existing qPCR assays are often not suitable and new primers have to be designed ( Hermann-Bank et al., 2013 Ishii, Segawa & Okabe, 2013 Kleyer, Tecon & Or, 2017). However, in order to work efficiently, high-throughput qPCR systems use identical PCR chemistry and PCR conditions for all reactions taking place on a single chip. High-throughput microfluidic qPCR brings further advantages including the fast generation of results, a lower cost per sample and fewer errors due to automatic distribution of samples and reagents. Culture-based diagnostic methods are progressively being replaced by qPCR due to advantages such as faster results, more specific detection, and the ability to detect sub-dominant populations ( Postollec et al., 2011). 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).
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