Rapid genotypic detection ofBacillus anthracisand theBacillus cereusgroup by multiplex real-time PCR melting curve analysis

Abstract

Bacillus anthracis has four plasmid possible virulence genotypes: pXO1+/pXO2+, pXO1+/pXO2-, pXO1-/pXO2+ or pXO1-/pXO2-. Due to the lack of a specific chromosomal marker for B. anthracis, differentiation of the pXO1-/pXO2- form of B. anthracis from closely related Bacillus cereus group species is difficult. In this study, we evaluate the ability of sspE, pXO1 and pXO2 primers to discriminate individual B. anthracis and the B. cereus group genotypes using multiplex real-time PCR and melting curve analysis. Optimal conditions for successful multiplex assays have been established. Purified DNAs from 38 bacterial strains including 11 strains of B. anthracis and 18 B. cereus group strains were analyzed. Nine of the B. cereus group near-neighbor strains were shown by multilocus sequence typing to be phylogenetically proximate to the B. anthracis clade. We have demonstrated that the four plasmid genotypes of B. anthracis and B. cereus group near-neighbors were differentially and simultaneously discriminated by this assay.

Implications of Limits of Detection of Various Methods for Bacillus anthracis in Computing Risks to Human Health

“…Considering the median instrument limit of detection, realtime PCR and PCR were the most sensitive methods, with median instrument limits of detection of 430 and 440 cells/ml, respectively. It should be noted that there was one instrument limit of detection (4.29 ϫ 10 6 cells/ml) that was not added to the distribution for real-time PCR because it was a multiplex assay, and the other instrument limits of detection in the distribution were from a singleplex assay (42). The least-sensitive methods were Raman spectroscopy and mass spectrometry, with median instrument limits of detection of approximately 1.0 ϫ 10 7 and 8.0 ϫ 10 7 cells/ml, respectively.…”

In silico and in vitro evaluation of PCR-based assays for the detection ofBacillus anthracischromosomal signature sequences

“…30 These genetic markers provide limited specificity and require additional timeconsuming and labor-intensive post-PCR analysis steps. Other areas of the chromosome have also been investigated as potential DNA-targets for identification purposes, including the so-called BA813 [31][32][33][34][35][36][37][38] and BA5510 sequences, 19 genes bclB, 39 sap, 40,41 saspB, 5,42 and sspE, 22,43 the B-type small acid-soluble spore protein gene (SASP), 44 a glycosyltransferase group 1 family protein, 45 a protein showing similarities with an abhydrolase, 18 and several DNA loci located on prophage regions, 17 i.e., BA5345, 21 BA5357, 46 and PL3. 47 Although most of these regions have been claimed to be anthrax-specific, B. cereus strains sometimes yield false positive results.…”

The Potential of High Resolution Melting Analysis (Hrma) to Streamline, Facilitate and Enrich Routine Diagnostics in Medical Microbiology

“…Makinen et al 45 focused on Bordetella pertusis with the aim of rapid gene typing of gene variants of the pertussis toxin using hybridization probes melting curve analysis. In order to obtain a reliable tool for detection of Bacillus anthracis and B. cereus, Kim et al 46 developed a multiplex real-time PCR melting curve analysis. A high degree of relatedness was demonstrated by several methods.…”

“…The discrimination among B. anthracis and B. cereus is then difficult. The melting analysis assay was able to provide a rapid, inexpensive and sensitive tool to simultaneously detect, from purified genomic DNA, all B. anthracis virulence genotypes and near-neighbor B. cereus group chromosomes 46 . De Medici et al 47 performed a specific reaction with primers targeting the region coding for a major subunit protein of a fimbrial structure on the surface of Salmonella enterica serotype Enteritidis.…”