Lista Florigio, Faggioni Giovanni, Valjevac Samina, Ciammaruconi Andrea, Vaissaire Josée, le Doujet Claudine, Gorgé Olivier, De Santis Riccardo, Carattoli Alessandra, Ciervo Alessandra, Fasanella Antonio, Orsini Francesco, D'Amelio Raffaele, Pourcel Christine, Cassone Antonio, Vergnaud Gilles
Army Medical Research Center, Via Santo Stefano Rotondo 4 00184 Rome (Italy).
BMC Microbiol. 2006 Apr 6;6:33. doi: 10.1186/1471-2180-6-33.
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.
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.
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.
炭疽病的病原体炭疽芽孢杆菌的基因组高度单一，这使得菌株之间的区分变得困难。此前已描述了一种基于20个标记的多位点可变数目串联重复序列（VNTR）分析（MLVA）检测方法。它具有相当强的鉴别力、可重复性且成本低，特别是因为所提出的标记可以通过琼脂糖凝胶电泳进行分型。然而，在紧急情况下，需要更快的基因分型以及访问代表性数据库。
使用一种25个位点的MLVA检测方法对炭疽芽孢杆菌参考菌株以及来自法国和意大利的分离株进行基因分型，该方法结合了21个先前描述的位点和4个新位点。DNA在4个多重PCR反应中进行扩增，所得25个扩增子的长度通过自动毛细管电泳进行估计。结果具有可重复性，并且一旦考虑到迁移模式的差异，数据与其他基于凝胶的方法一致。一些先前通过琼脂糖凝胶电泳无法分辨的等位基因可以通过毛细管电泳分辨出来，从而进一步提高了检测分辨率。一个特定的位点Bams30是27 bp串联重复序列和9 bp串联重复序列之间重组的结果。对阵列的分析说明了串联重复序列的进化过程。
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问题，2001年炭疽信件事件就说明了这一点。在本报告中，我们描述了先前发表的用于炭疽芽孢杆菌基因分型的MLVA方法的升级，并将该方法应用于法国和意大利炭疽芽孢杆菌菌株集合的分型。与仅使用8个位点的报告相比，所研究的标记数量增加极大地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，并提出了两个新分支D和E。由于在此实现的升级，现在可以通过自动毛细管电泳或更易操作但速度较慢且对某些标记稍欠准确的琼脂糖凝胶方法进行精确的基因分型。
