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

2020; Nature Portfolio; Volume: 10; Issue: 1 Linguagem: Inglês

10.1038/s41598-020-57742-z

2045-2322

Matteo Perini, Aurora Piazza, Simona Panelli, Domenico Di Carlo, Marta Corbella, Floriana Gona, Francesca Vailati, P. Marone, Daniela María Cirillo, Claudio Farina, Gian Vincenzo Zuccotti, Francesco Comandatore,

Bacteriophages and microbial interactions

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.