Identification of capsule-forming Bacillus anthracis spores with the PCR and a novel dual-probe hybridization format

Abstract

Anthrax is a fatal infection of humans and livestock that is caused by the gram-positive bacterium Bacillus anthracis. The virulent strains of B. anthracis are encapsulated and toxigenic. In this paper we describe the development of a PCR technique for identifying spores of B. anthracis. Two 20-mer oligonucleotide primers specific for the capB region of 60-MDa plasmid pXO2 were used for amplification. The amplification products were detected by using biotin-and fluorescein-labeled probes in a novel dual-probe hybridization format. Using the combination of PCR amplification and dual-probe hybridization, we detected two copies of the bacterial genome. Because the PCR assay could detect a minimum of 100 unprocessed spores per PCR mixture, we attempted to facilitate the release of DNA by comparing the effect of limited spore germination with the effect of mechanical spore disruption prior to PCR amplification. The two methods were equally effective and allowed us to identify single spores of B. anthracis in PCR mixtures.

Polymerase chain reaction-ELISA to detect Bacillus anthracis from soil samples-limitations of present published primers

“…The primers used for the detection of the capsule-encoding region of the plasmid pX02 and the protective antigen gene of the plasmid pX01 have been published by Makino et al (1993), Johns et al (1994), Reif et al (1994), Beyer et al (1995), Sjöstedt et al (1995Sjöstedt et al ( , 1997 and Ramisse et al (1996). For detection of the chromosome the primers published by Patra et al (1996) were used.…”

“…Recently, some successful attempts were made to detect a single B. anthracis spore by the polymerase chain reaction (PCR) (Titball et al 1991;Carl et al 1992;Makino et al 1993;Johns et al 1994;Reif et al 1994;Sjöstedt et al 1995Sjöstedt et al , 1997Patra et al 1996;Ramisse et al 1996). Beyer et al (1995) published a nested PCR method coupled with a non-selective pre-enrichment step for the detection of less than 10 spores of B. anthracis from 100 g soil.…”

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

“…anthracis is a highly fatal infectious agent in animals and humans and therefore its early and unambiguous diagnostic detection is essential for successful treatment and disease prevention. There have been many efforts to utilize rapid DNA-based detection methods, such as PCR [22][23][24][25][26][27][28][29][30][31][32][33], to replace time-consuming biochemical or culture-based diagnostic tests [34]. PCR-based methods can readily differentiate vaccine or fully virulent B. anthracis plasmid genotypes [22][23][24]26,35].…”

“…There have been many efforts to utilize rapid DNA-based detection methods, such as PCR [22][23][24][25][26][27][28][29][30][31][32][33], to replace time-consuming biochemical or culture-based diagnostic tests [34]. PCR-based methods can readily differentiate vaccine or fully virulent B. anthracis plasmid genotypes [22][23][24]26,35]. However, plasmid-cured B. anthracis [36][37][38][39], or near-neighbor species containing B. anthracis closely-related plasmids [40], are very difficult to distinguish from B. anthracis.…”

Isolation of a specific chromosomic DNA sequence of Bacillus anthracis and its possible use in diagnosis

“…We did not yet apply our assay directly to the spores, however, since DNA can be efficiently released from spores of B. anthrucis using bead beating, germination, heat shocking or a combination of these techniques [24], the PCR method described here could be used with spores. Since both the capsule and the exotoxin are essential virulence factors for B. anthracis, the oligonucleotides designed in this study together with other primers specific for capsule and toxin genes [21,23,25,26] will be useful for a more in-depth survey [27]. This method can be applied to perform a rapid screening directly with a suspected colony picked on a plate.…”