Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages.
Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages.
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DOI：
10.1371/journal.pone.0041253
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Gagneux S
中科院分区：
文献类型：
--
作者：
Stucki D;Malla B;Hostettler S;Huna T;Feldmann J;Yeboah-Manu D;Borrell S;Fenner L;Comas I;Coscollà M;Gagneux S
越来越多的证据表明，结核分枝杆菌复合群（MTBC）的菌株变异可能影响结核病感染和疾病的结局。为了评估基因型-表型关联，需要遗传学上稳健的分子标记和适当的基因分型工具。目前大多数MTBC的基因分型方法是基于移动的或重复的DNA元件。由于这些元件易于趋同进化，因此相应的基因分型技术对于系统发育研究和菌株分类是次优的。相比之下，单核苷酸多态性（SNP）是将MTBC分类为系统发生谱系的理想标记，因为它们表现出非常低的同源性。在这项研究中，我们开发了两个互补的SNP为基础的基因分型方法来分类菌株到六个主要的人类相关的谱系的MTBC，“北京”亚系，分支包括牛分枝杆菌和分枝杆菌capillary。从22个MTBC全基因组序列中获得系统发育信息SNP。第一种测定法，称为MOL-PCR，是一种连接依赖性PCR，通过荧光微球和Luminex流式细胞仪进行信号检测，同时询问8个SNP。第二种测定法基于用于单重SNP分型的六种单独TaqMan实时PCR测定法。我们比较了两组临床MTBC分离株的MOL-PCR和TaqMan结果。这两种方法完全同意时，分配36个特点鲜明的菌株到主要的系统发育谱系。MOL-PCR和TaqMan检测等位基因的灵敏度分别为98.6%和98.8%。另外一组78株未知临床分离株的分型显示，等位基因调用的灵敏度分别为99.2%和100%，两种方法之间的谱系分配的一致性为100%。虽然MOL-PCR和TaqMan都具有高度的敏感性和特异性，但MOL-PCR对于没有先前信息的分离株的分类是理想的，而TaqMan用于确认更快。此外，这两种方法都是快速，灵活和廉价的。
There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the “Beijing” sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive.
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影响因子：
9.4
作者：
Bouakaze, C.;Keyser, C.;Ludes, B.
通讯作者：
Ludes, B.
影响因子：
9.4
作者：
de los Monteros, LEE;Galán, JC;Blázquez, J
通讯作者：
Blázquez, J
影响因子：
3.2
作者：
Filliol, I;Motiwala, AS;Alland, D
通讯作者：
Alland, D
影响因子：
2.2
作者：
Deshpande, Alina;Gans, Jason;White, P. Scott
通讯作者：
White, P. Scott
DOI：
10.1016/j.ddmec.2010.09.004
发表时间：
2010
期刊：
Drug discovery today. Disease mechanisms
影响因子：
--
作者：
Coscolla, Mireilla;Gagneux, Sebastien
通讯作者：
Gagneux, Sebastien