Specific detection of Bacillus anthracis using aTaqMan® mismatch amplification mutation assay

Abstract

Single nucleotide polymorphisms (SNPs) are increasingly recognized as important diagnostic markers for the detection and differentiation ofBacillus anthracis. The use ofSNP markers for identifying B. anthracis DNA in environmental samples containing genetically similar bacteria requires the ability to amplify and detect DNA with single nucleotide specificity. We designed a TaqMan® mismatch amplification mutation assay (TaqMAMA) around a SNP in the plcR gene ofB. anthracis. The assay permits specific, low-level detection (25fg DNA) of this B. anthracis-specific SNP, even in the presence of environmental DNA extracts containing a 20,000-fold excess of the alternate allele. We anticipate that the ability to selectively amplify and detect low copy number DNAs with single nucleotide specificity will represent a valuable tool in the arena ofbiodefense and microbialforensics.