Abstract
In this work we present EasyPrimer, a user-friendly online tool developed to assist pan-PCR and High Resolution Melting (HRM) primer design. The tool finds the most suitable regions for primer design in a gene alignment and returns a clear graphical representation of their positions on the consensus sequence. EasyPrimer is particularly useful in difficult contexts, e.g. on gene alignments of hundreds of sequences and/or on highly variable genes. HRM analysis is an emerging method for fast and cost saving bacterial typing and an HRM scheme of six primer pairs on five Multi-Locus Sequence Type (MLST) genes is already available for Klebsiella pneumoniae. We validated the tool designing a scheme of two HRM primer pairs on the hypervariable gene wzi of Klebsiella pneumoniae and compared the two schemes. The wzi scheme resulted to have a discriminatory power comparable to the HRM MLST scheme, using only one third of primer pairs. Then we successfully used the wzi HRM primer scheme to reconstruct a Klebsiella pneumoniae nosocomial outbreak in few hours. The use of hypervariable genes reduces the number of HRM primer pairs required for bacterial typing allowing to perform cost saving, large-scale surveillance programs.
Highlights
Most methods used for the identification and typing of prokaryotes are based on DNA amplification and sequencing
EasyPrimer is a user-friendly open-source tool developed to assists primer design in difficult contexts, e.g. on an alignment of hundreds of sequences and/or on hypervariable genes
The tool uses as input a nucleotide multi-fasta file and identifies the best regions for primer design: two low variable regions flanking a highly variable one
Summary
Most methods used for the identification and typing of prokaryotes are based on DNA amplification and sequencing. A nosocomial outbreak occurs when the number of patients infected by a pathogen increases above the expected in a limited time[2] In these situations, it is fundamental to determine the clonality of bacteria causing disease in the patients to define the proper strategy to handle the emergency. HRM is a single-step procedure for the discrimination of sequence variants on the basis of their melting temperature. This method allows to perform bacterial typing in less than five hours[7]. MinimumSNP identifies single informative positions, not necessarily grouped in a single portion of the gene In other words, it does not indicate which regions are more suitable for primer design: two low-variable regions neighbouring a SNP-rich informative stretch. The user of MinimumSNP has to choose one (or few) SNPs and design primers around it (or around them)
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