A new Bacillus anthracis specific gene and its diagnostic application

Abstract

Introduction: B. anthracis is a highly fatal infectious agent in animals and humans. In this era of bioterrorism, the risk of exposing a large population to this lethal pathogen has increased dramatically. Therefore, its early and accurate diagnostic detection is essential for successful treatment and control of spread. Due to the isolation of very closely related Bacillus cereus group species, definitive molecular identification of Bacillus anthracis needs detection of specific markers for at least three different loci, one chromosome and two virulence plasmids (pXO1, and pXO2). It is difficult to find a chromosome-specific marker due to the genetic similarity among the B. cereus group species. There are several reports of the use of a B. anthraics chromosome-specific marker, but there has not been a marker which was extensively tested in vitro with many strains of the B. cereus group species, including the closely related ones to B. anthracis. In addition, most of currently reported multiplex real-time PCR methods rely on the use of expensive multiple probes to simultaneously detect multiple markers. Here, I introduce a new specific chromosomal marker (BA1698) for B. anthracis, the specificity of which was extensively tested with a lot of bacterial strain and a highly sensitive non-probe (SYBR green)-based multiplex real-time PCR method that identifies up to four products formed together in a single reaction tube. I optimally designed it for a sensitive multiplex melting curve analysis for the differential co-detection of the two plasmids, a chromosomal marker (BA1698) and 16S rRNA gene as an internal control. Methods: BA1698 was found as a candidate for a specific chromosomal marker of B. anthracis during genome-wide screening for B. anthracis chromosome specific genes, which was processed by comparative analysis of all putative open reading frames (ORFs) of B. anthracis Ames strain with the genome sequences of several B. cereus group strains. SYBR green-based multiplex real-time PCR method was developed for rapid and accurate molecular diagnosis of B. anthracis by designing the primers, in-house reaction mastermix and cycle conditions. The specificity of this method was tested with 332 strains of bacteria including 11 genomic DNA of B. anthracis, seven of the most closely related neighbors of B. anthracis, 27 closely related B. cereus group strains, 250 (158 known and 92 probable) strains of B. cereus group species. The limit of detection was determined by probit analysis. Results: The SYBR green-based multiplex real-time PCR clearly and specifically identified all the target markers (BA1698, pXO1, pXO2, and 16S rRNA gene) among 332 strains of bacteria. The primer specificity for the chromosomal marker was further confirmed by in silico analysis using BLAST algorithm. The limit of detection determined by probit analysis was 15.7 copies (~100 fg of DNA) per reaction (95% confidence interval, 11.5 – 27.4 copies) which was comparable to that of multiprobe-based methods Conclusions: In this study, a specific chromosomal marker for B. anthracis was discovered. The high specificity was extensively verified by molecular method. Including this marker, a sensitive SYBR green-based multiplex real-time PCR assay has been developed, which is rapid, simple and sensitive that it can be used as an alternative molecular diagnostic tool for the specific detection of B. anthracis.