Genotyping of Bacillus anthracis strains based on automated capillary 25-loci multiple locus variable-number tandem repeats analysis
Genotyping of Bacillus anthracis strains based on automated capillary 25-loci multiple locus variable-number tandem repeats analysis
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DOI：
10.1186/1471-2180-6-33
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发表时间：
2006-04-06
期刊：
影响因子：
4.2
通讯作者：
Vergnaud, Gilles
中科院分区：
文献类型：
--
作者：
Lista, Florigio;Faggioni, Giovanni;Vergnaud, Gilles
背景：炭疽病原菌炭疽芽孢杆菌的基因组高度单态，很难区分不同菌株。本文介绍了一种基于20个标记的多基因座可变数目串联重复序列分析方法(MLVA)。它具有相当大的识别力、重复性和低成本，特别是因为所提出的标记可以用琼脂糖凝胶电泳法进行分型。但在紧急情况下，更快的基因分型和访问代表性数据库是必要的。结果：用25个座位的MLVA方法结合21个先前描述的座位和4个新的座位对来自法国和意大利的炭疽参考菌株和分离株进行了基因分型。在4个多重PCR反应中扩增DNA，并用自动毛细管电泳法估计得到的25个扩增片段的长度。如果考虑到运动模式的差异，结果是可重复性的，并且数据与其他基于凝胶的方法一致。一些先前未经琼脂糖凝胶电泳法分离的等位基因可用毛细管电泳法分离，从而进一步提高分析分辨率。一个特殊的基因座Bams30是27个碱基的串联重复序列和9个碱基的串联重复序列重组的结果。对序列的分析说明了串联重复的进化过程。结论：在疑似生物恐怖主义的危机情况下，标准化、速度和准确性以及参考分型数据的可用性是重要的问题，2001年的炭疽字母事件说明了这一点。在这份报告中，我们描述了先前发表的用于炭疽杆菌基因分型的MLVA方法的升级，并将该方法应用于法国和意大利炭疽杆菌株系的分型。与仅使用8个基因座的报告相比，研究的标记数量增加，大大提高了该技术的鉴别力。描述了一株属于B分支的意大利菌株，并提出了D和E两个新分支。由于这里实现了升级，现在可以通过自动毛细管电泳法或更容易获得但速度较慢的琼脂糖凝胶方法进行精确的基因分型。
Background: The genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary.Results: Genotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats.Conclusion: In a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the B branch was described, and two new branches, D and E, are proposed. Owing to the upgrading achieved here, precise genotyping can now be produced either by automated capillary electrophoresis, or by the more accessible but slower and for some markers slightly less accurate agarose gel methodology.