Strain-specific single-nucleotide polymorphism assays for the Bacillus anthracis Ames strain
Strain-specific single-nucleotide polymorphism assays for the Bacillus anthracis Ames strain
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DOI：
10.1128/jcm.01233-06
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发表时间：
2007-01-01
影响因子：
9.4
通讯作者：
Keim, Paul
中科院分区：
文献类型：
--
作者：
Van Ert, Matthew N.;Easterday, W. Ryan;Keim, Paul
通过进化分析和对基因组序列的详尽检查相结合，可以开发出高精度的诊断和法医检测方法。在炭疽芽孢杆菌中，全基因组测序工作揭示了8个不同菌株之间约3,500个单核苷酸多态(SNPs)，进化分析提供了典型SNPs的鉴定。我们以前已经证明SNPs在进化上是高度稳定的，炭疽杆菌的克隆性质使它们成为这种病原体亚型的理想标志。在这里，我们识别了定义炭疽杆菌谱系的SNPs，其中包含Ames菌株，2001年美国生物恐怖袭击中使用的菌株。通过测序和实时定量聚合酶链式反应来验证这些SNPs在炭疽杆菌菌株中的有效性，包括(I)88个全球和遗传多样性的菌株；(Ii)经多位点可变数目串联重复分析(MLVA)证明与Ames菌株有遗传亲缘关系的菌株；以及(Iii)Ames菌株的几个不同的实验室种群，包括来自2001年字母攻击的临床菌株。发现6个SNP对Ames菌株高度特异；4个在染色体上，1个在pX01质粒上，1个在pX02质粒上。所有6个SNP都将炭疽杆菌Ames菌株与88个独特的炭疽杆菌菌株区分开来，而6个SNP中有5个将Ames与其近亲区分开来。将这些SNPs与实时聚合酶链式反应结合使用，可以对Ames菌株进行特异和灵敏的(100 fg模板DNA)鉴定。这种基于进化和基因组学的方法为发现炭疽杆菌菌株特异性SNPs提供了一种有效的手段。
Highly precise diagnostics and forensic assays can be developed through a combination of evolutionary analysis and the exhaustive examination of genomic sequences. In Bacillus anthracis, whole-genome sequencing efforts revealed ca. 3,500 single-nucleotide polymorphisms (SNPs) among eight different strains and evolutionary analysis provides the identification of canonical SNPs. We have previously shown that SNPs are highly evolutionarily stable, and the clonal nature of B. anthracis makes them ideal signatures for subtyping this pathogen. Here we identified SNPs that define the lineage of B. anthracis that contains the Ames strain, the strain used in the 2001 bioterrorist attacks in the United States. Sequencing and real-time PCR were used to validate these SNPs across B. anthracis strains, including (i) 88 globally and genetically diverse isolates; (ii) isolates that were shown to be genetic relatives of the Ames strain by multiple-locus variable number tandem repeat analysis (MLVA); and (iii) several different lab stocks of the Ames strain, including a clinical isolate from the 2001 letter attack. Six SNPs were found to be highly specific for the Ames strain; four on the chromosome, one on the pX01 plasmid, and one on the pX02 plasmid. All six SNPs differentiated the B. anthracis Ames strain from the 88 unique B. anthracis strains, while five of the six separated Ames from its close genetic relatives. The use of these SNPs coupled with real-time PCR allows specific and sensitive (< 100 fg of template DNA) identification of the Ames strain. This evolutionary and genomics-based approach provides an effective means for the discovery of strain-specific SNPs in B. anthracis.