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Accepted on ,

Technical Briefs

Optimization of High-Resolution Melting Analysis for Low-Cost and Rapid Screening of Allelic Variants of Bacillus anthracis by Multiple-Locus Variable-Number Tandem Repeat Analysis,

¹ Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy
² Molecular Biology Section, Army Medical and Veterinary Research Center, Rome, Italy
³ Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata-Anthrax Reference Institute of Italy, Foggia, Italy
⁴ Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Rome, Italy
⁵ Cattedra di Allergologia e Immunologia Clinica, II Facoltà di Medicina, Università di Roma "La Sapienza", Rome, Italy, and Direzione Generale della Sanità Militare, Rome, Italy

^* To whom correspondence should be addressed. E-mail: alecara{at}iss.it.

Background: 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.

Methods: 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.

Results: 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.

Conclusions: The described HRMA application for screening B. anthracis VNTR loci is fast and widely accessible and may prove particularly useful under field conditions.