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

Sci Rep. 2020 Jan 28;10(1):1307. doi: 10.1038/s41598-020-57742-z.

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.

MeSH terms

  • Bacterial Outer Membrane Proteins / genetics*
  • DNA Primers*
  • Genome, Bacterial
  • Genomics / methods
  • Klebsiella Infections / diagnosis*
  • Klebsiella Infections / microbiology*
  • Klebsiella pneumoniae / classification*
  • Klebsiella pneumoniae / genetics*
  • Phylogeny
  • Polymerase Chain Reaction / methods
  • Software*

Substances

  • Bacterial Outer Membrane Proteins
  • DNA Primers