Fortini Daniela, Ciammaruconi Andrea, De Santis Riccardo, Fasanella Antonio, Battisti Antonio, D'Amelio Raffaele, Lista Florigio, Cassone Antonio, Carattoli Alessandra
Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy.
Clin Chem. 2007 Jul;53(7):1377-80. doi: 10.1373/clinchem.2007.085993. Epub 2007 May 24.
Molecular genotyping of Bacillus anthracis, the etiologic agent of anthrax, is important for differentiating and identifying strains from different geographic areas and for tracing strains deliberately released in a bioterrorism attack. We previously described a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) based on 25 marker loci. Although the method has great differentiating power and reproducibility, faster genotyping at low cost may be requested to accurately identify B. anthracis strains in the field.
We used the High Resolution Melter-1 (Idaho Technology) and a saturating dye of double-stranded DNA (LCGreen I) to identify alleles via PCR and melting-curve analysis of the amplicons. We applied high-resolution melting analysis (HRMA) to a collection of 19 B. anthracis strains.
HRMA produced reproducible results for 6 of the 25 B. anthracis loci tested. These easily interpretable and distinguishable melting curve results were consistent with MLVA results obtained for the same alleles. The feasibility of this method was demonstrated in testing of different allelic variants for the 6 selected loci.
The described HRMA application for screening B. anthracis VNTR loci is fast and widely accessible and may prove particularly useful under field conditions.
炭疽的病原体炭疽芽孢杆菌的分子基因分型对于区分和鉴定来自不同地理区域的菌株以及追踪生物恐怖袭击中故意释放的菌株非常重要。我们之前描述了一种基于25个标记位点的多位点可变数目串联重复序列（VNTR）分析（MLVA）方法。尽管该方法具有很强的区分能力和可重复性，但可能需要以低成本进行更快的基因分型，以便在现场准确鉴定炭疽芽孢杆菌菌株。
我们使用高分辨率熔解仪-1（爱达荷技术公司）和双链DNA饱和染料（LCGreen I），通过对扩增子进行PCR和熔解曲线分析来鉴定等位基因。我们将高分辨率熔解分析（HRMA）应用于19株炭疽芽孢杆菌菌株。
对于所测试的25个炭疽芽孢杆菌位点中的6个，HRMA产生了可重复的结果。这些易于解释和区分的熔解曲线结果与相同等位基因的MLVA结果一致。该方法在测试所选6个位点的不同等位基因变体时的可行性得到了证明。
所描述的用于筛选炭疽芽孢杆菌VNTR位点的HRMA应用快速且广泛适用，在现场条件下可能特别有用。
