Groupe hospitalier Chenevier-Mondor, APHP, Laboratoire de Parasitologie-Mycologie, Créteil, France.
J Microbiol Methods. 2011 Sep;86(3):357-63. doi: 10.1016/j.mimet.2011.06.017. Epub 2011 Jul 1.
Genetic studies of the protozoan parasite Toxoplasma gondii have identified three main distinct types according to virulence in some hosts. Several methods have been developed to differentiate genotypes currently dominated by microsatellite markers targeting single-copy loci. We analyzed the possibility of using the 35-fold repetitive B1 gene via high-resolution melting (HRM) curve analysis. Sequencing of the B1 gene of 14 reference strains (four Type I, six Type II, and four Type III strains) identified 18 single nucleotide polymorphisms (SNP). Primers were designed to amplify eight of them for HRM analysis and for relative quantification of each nucleotide variation using SNaPshot mini-sequencing. Genotyping with five microsatellite markers was performed for comparison. Two to four HRM profiles were obtained depending on the SNP tested. The differences observed relied on the different ratios of nucleotides at the SNP locus as evidenced via SNaPshot mini-sequencing. The three main lineages could be distinguished by using several HRM profiles. Some HRM profiles proved more informative than the analysis based on five microsatellite markers, showing additional differences in Type I and Type II strains. Using HRM analysis, we obtained at least an equally good discrimination of the main lineages than that based on five microsatellite markers.
对原生动物寄生虫弓形虫的遗传研究根据在某些宿主中的毒力确定了三种主要的不同类型。已经开发了几种方法来区分基因型，目前主要使用针对单拷贝基因座的微卫星标记。我们通过高分辨率熔解（HRM）曲线分析分析了使用 35 倍重复 B1 基因的可能性。对 14 个参考菌株（4 个 I 型、6 个 II 型和 4 个 III 型菌株）的 B1 基因测序确定了 18 个单核苷酸多态性（SNP）。设计了引物来扩增其中的 8 个用于 HRM 分析，以及使用 SNaPshot 迷你测序法对每个核苷酸变异进行相对定量。进行了 5 个微卫星标记的基因分型比较。根据测试的 SNP，获得了 2 到 4 种 HRM 图谱。观察到的差异取决于 SNP 基因座中核苷酸的不同比例，这通过 SNaPshot 迷你测序得到证实。可以通过使用几种 HRM 图谱来区分这三个主要谱系。一些 HRM 图谱比基于五个微卫星标记的分析提供了更多的信息，在 I 型和 II 型菌株中显示了额外的差异。使用 HRM 分析，我们获得了与基于五个微卫星标记的分析至少同样好的主要谱系的区分。
