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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.

EasyPrimer: user-friendly tool for pan-PCR/HRM primers design. Development of an HRM protocol on wzi gene for fast Klebsiella pneumoniae typing