Use of high-resolution melting and melting temperature-shift assays for specific detection and identification of Bacillus anthracis based on single nucleotide discrimination

Abstract

Single nucleotide polymorphisms (SNPs) are important diagnostic markers for the detection and differentiation of Bacillus anthracis. High-Resolution Melting (HRM) and Melting Temperature (Tm)-shift methods are two approaches that enable SNP detection without the need for expensive labeled probes. We evaluated the potential diagnostic capability of those methods to discriminate B. anthracis from the other members of the B. cereus group. Two assays targeting B. anthracis-specific SNPs in the plcR and gyrA genes were designed for each method and used to genotype a panel of 155 Bacilli strains. All B. anthracis isolates (n=65) were correctly and unambiguously identified. Assays also proved to be appropriate for the direct genotyping of biological samples. They could reliably detect B. anthracis in contaminated organs containing as little as 10(3)CFU/ml, corresponding to a few genome equivalents per reaction. The HRM and Tm-shift applications described here represent valuable tools for specific identification of B. anthracis at reduced cost.