title: A novel multiplex PCR discriminates Bacillus anthracis and its genetically related strains from other Bacillus cereus group species.
abstract: Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis.
authors.affiliation: Hokudai Center for Zoonosis Control in Zambia, School of Veterinary Medicine, The University of Zambia, Lusaka, Zambia; Department of Disease Control, School of Veterinary Medicine, The University of Zambia, Lusaka, Zambia.
authors.forename: Hirohito
authors.initials: H
authors.lastname: Ogawa
authors.affiliation: Division of Infection and Immunity, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Fujikura
authors.affiliation: Division of Infection and Immunity, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Ohnuma
authors.affiliation: Division of Infection and Immunity, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan.
authors.forename: Naomi
authors.initials: N
authors.lastname: Ohnishi
authors.affiliation: Department of Paraclinical Studies, School of Veterinary Medicine, The University of Zambia, Lusaka, Zambia.
authors.forename: Bernard M
authors.initials: BM
authors.lastname: Hang'ombe
authors.affiliation: Division of Bacteriology, Department of Infectious Diseases Control, International Research Center for Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Pathogenic Microbes Repository Unit, International Research Center for Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Mimuro
authors.affiliation: Department of Microbiology, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Gifu, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ezaki
authors.affiliation: Department of Disease Control, School of Veterinary Medicine, The University of Zambia, Lusaka, Zambia.
authors.forename: Aaron S
authors.initials: AS
authors.lastname: Mweene
authors.affiliation: Hokudai Center for Zoonosis Control in Zambia, School of Veterinary Medicine, The University of Zambia, Lusaka, Zambia; Department of Disease Control, School of Veterinary Medicine, The University of Zambia, Lusaka, Zambia; Division of Infection and Immunity, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan; Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Higashi
mesh_terms: D000820:Animal Diseases / Q000175:diagnosis / Q000382:microbiology; D000818:Animals; D000881:Anthrax / Q000175:diagnosis / Q000382:microbiology; D001408:Bacillus anthracis / Q000145:classification / Q000235:genetics*; D001409:Bacillus cereus / Q000145:classification / Q000235:genetics*; D002876:Chromosomes, Bacterial; D016908:Gram-Positive Bacterial Infections / Q000175:diagnosis / Q000382:microbiology; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D010957:Plasmids / Q000235:genetics; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S