Pasala Ajay Ratan, Perini Matteo, Piazza Aurora, Panelli Simona, Di Carlo Domenico, Loretelli Cristian, Cafiso Alessandra, Inglese Sonia, Gona Floriana, Cirillo Daniela Maria, Zuccotti Gian Vincenzo, Comandatore Francesco
Department of Biomedical and Clinical Sciences "L. Sacco", Università di Milano, Pediatric Clinical Research Center "Romeo and Enrica Invernizzi", Milan, Italy.
Department of Clinical Surgical Diagnostic and Pediatric Sciences, Microbiology and Clinical Microbiology Unit, University of Pavia, Pavia, Italy.
AMB Express. 2020 Dec 14;10(1):217. doi: 10.1186/s13568-020-01164-7.
High resolution melting (HRM) is a fast closed-tube method for nucleotide variant scanning applicable for bacterial species identification or molecular typing. Recently a novel HRM-based method for Klebsiella pneumoniae typing has been proposed: it consists of an HRM protocol designed on the capsular wzi gene and an HRM-based algorithm of strains clustering. In this study, we evaluated the repeatability and reproducibility of this method by performing the HRM typing of a set of K. pneumoniae strains, on three different instruments and by two different operators. The results showed that operators do not affect melting temperatures while different instruments can. Despite this, we found that strain clustering analysis, performed using MeltingPlot separately on the data from the three instruments, remains almost perfectly consistent. The HRM method under study resulted highly repeatable and thus reliable for large studies, even when several operators are involved. Furthermore, the HRM clusters obtained from the three different instruments were highly conserved, suggesting that this method could be applied in multicenter studies, even if different instruments are used.
高分辨率熔解曲线分析（HRM）是一种快速的闭管核苷酸变异扫描方法，适用于细菌物种鉴定或分子分型。最近，一种基于HRM的新型肺炎克雷伯菌分型方法被提出：它由基于荚膜wzi基因设计的HRM方案和基于HRM的菌株聚类算法组成。在本研究中，我们通过在三种不同仪器上由两名不同操作人员对一组肺炎克雷伯菌菌株进行HRM分型，评估了该方法的重复性和再现性。结果表明，操作人员不会影响熔解温度，而不同仪器会有影响。尽管如此，我们发现，使用MeltingPlot分别对来自三种仪器的数据进行菌株聚类分析，结果几乎完全一致。所研究的HRM方法具有高度重复性，因此即使涉及多个操作人员，对于大型研究也是可靠的。此外，从三种不同仪器获得的HRM聚类高度保守，这表明即使使用不同仪器，该方法也可应用于多中心研究。
