Department of Biomedical and Clinical Sciences "L. Sacco", Università di Milano, Pediatric Clinical Research Center "Romeo and Enrica Invernizzi", Milan, 20157, Italy.
S.C. Microbiologia e Virologia, Fondazione IRCCS Policlinico San Matteo, Pavia, 27100, Italy.
Sci Rep. 2020 Jan 28;10(1):1307. doi: 10.1038/s41598-020-57742-z.
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.
在这项工作中，我们介绍了 EasyPrimer，这是一个用户友好的在线工具，旨在辅助 pan-PCR 和高分辨率熔解（HRM）引物设计。该工具在基因比对中找到最适合引物设计的区域，并返回其在共识序列上位置的清晰图形表示。EasyPrimer 在困难的情况下特别有用，例如在数百个序列的基因比对和/或高度可变的基因上。HRM 分析是一种用于快速和节省成本的细菌分型的新兴方法，已经有针对肺炎克雷伯菌的五个多位点序列分型（MLST）基因的六个引物对的 HRM 方案。我们通过设计肺炎克雷伯菌超变基因 wzi 的两个 HRM 引物对方案来验证该工具，并比较了这两个方案。wzi 方案的区分能力与 HRM MLST 方案相当，仅使用三分之一的引物对。然后，我们成功地在数小时内使用 wzi HRM 引物方案重建了一起医院获得性肺炎克雷伯菌暴发。使用高度可变基因减少了用于细菌分型的 HRM 引物对的数量，允许进行节省成本、大规模的监测计划。
