Brazilian Reference Center on Yersinia spp. other than Y. pestis, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, Ribeirão Preto, SP, Brazil.
J Microbiol Methods. 2012 Dec;91(3):329-35. doi: 10.1016/j.mimet.2012.09.006. Epub 2012 Oct 3.
Yersinia pseudotuberculosis is an enteric pathogen that is environmentally widespread and is known to cause human and animal infections. The development of a fast and inexpensive typing system is necessary to facilitate epidemiological studies of Y. pseudotuberculosis infections. In this study, we aimed to develop a method of Y. pseudotuberculosis genotyping based on determining differences in single-nucleotide polymorphisms (SNPs) using a high-resolution melting analysis (HRMA). Using a set of nine primer pairs, ten SNPs were screened from sequences in the 16S rRNA, glnA, gyrB and recA sequences of 12 Y. pseudotuberculosis strains that were deposited in the GenBank database. The genetic diversity of a collection of 40 clinical Y. pseudotuberculosis strains was determined using the HRMA method and the multilocus sequence typing (MLST) technique was used for comparison. Different melting profiles were found in five out of a total of nine analyzed fragments. A phylogenetic tree was constructed from the nucleotides that were identified in the nine analyzed fragments, and the tree demonstrated that Y. pseudotuberculosis strains were separated into two groups. The first cluster was composed of strains from the 1/O:1a serogroup and the second of strains from the 2/O:3 serogroup. The separation into two clusters based on distinct bio-serogroups of Y. pseudotuberculosis was consistent with the results in the MLST database. The simple and highly reproducible HRMA assay developed by us may be used as a rapid and cost-effective method to genotype Y. pseudotuberculosis strains of O:1 and O:3 serogroups and it can complement sequence-based methods facilitating epidemiological studies of this Yersinia species.
假结核耶尔森菌是一种肠道病原体，广泛存在于环境中，已知可引起人类和动物感染。开发一种快速且廉价的分型系统对于促进假结核耶尔森菌感染的流行病学研究是必要的。在本研究中，我们旨在开发一种基于高分辨率熔解分析（HRMA）确定单核苷酸多态性（SNP）差异的假结核耶尔森菌基因分型方法。使用一组九对引物，从 GenBank 数据库中存储的 12 株假结核耶尔森菌的 16S rRNA、glnA、gyrB 和 recA 序列中筛选出 10 个 SNP。使用 HRMA 方法确定了 40 株临床假结核耶尔森菌菌株的遗传多样性，并与多位点序列分型（MLST）技术进行了比较。在总共分析的 9 个片段中，有 5 个发现了不同的熔解曲线。从在 9 个分析片段中鉴定的核苷酸构建了一个系统发育树，该树表明假结核耶尔森菌菌株分为两组。第一组由 1/O:1a 血清群的菌株组成，第二组由 2/O:3 血清群的菌株组成。基于假结核耶尔森菌的不同生物血清群将其分为两组的结果与 MLST 数据库中的结果一致。我们开发的简单且高度可重复的 HRMA 测定法可用于快速且经济高效地对 O:1 和 O:3 血清群的假结核耶尔森菌菌株进行基因分型，并且可以补充基于序列的方法，促进对该耶尔森菌物种的流行病学研究。
