Genotyping of the protozoan pathogen Toxoplasma gondii using high-resolution melting analysis of the repeated B1 gene
Genotyping of the protozoan pathogen Toxoplasma gondii using high-resolution melting analysis of the repeated B1 gene
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DOI：
10.1016/j.mimet.2011.06.017
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发表时间：
2011-09-01
影响因子：
2.2
通讯作者：
Bretagne, Stephane
中科院分区：
文献类型：
--
作者：
Costa, Jean-Marc;Cabaret, Odile;Bretagne, Stephane
对原生动物弓形虫的遗传学研究根据某些宿主的毒力确定了三种主要的不同类型。目前已经开发了几种方法来区分目前以单拷贝基因座为靶标的微卫星标记所主导的基因类型。我们通过高分辨率熔融(HRM)曲线分析了利用35倍重复的B1基因的可能性。对14株参考菌株(4株I型、6株II型和4株I11型)的B1基因进行测序，鉴定出18个单核苷酸多态(SNP)。设计了用于扩增其中8个核苷酸的引物，用于HRM分析和使用快照微型测序对每个核苷酸变异进行相对量化。用5个微卫星标记进行基因分型比较。根据测试的SNP，获得了两到四个人力资源管理概况。观察到的差异依赖于SNP基因座核苷酸比例的不同，这一点通过快照微型测序得到了证明。通过使用几个HRM图谱可以区分三个主要的谱系。一些HRM图谱证明比基于五个微卫星标记的分析更具信息量，显示了类型II土地类型菌株的额外差异。利用HRM分析，我们至少获得了与基于5个微卫星标记的主要谱系同样好的判别力。(C)2011爱思唯尔B.V.保留所有权利。
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 Ill 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 land 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. (C) 2011 Elsevier B.V. All rights reserved.