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

A 277-bp long DNA fragment, Ba813, was isolated from an avirulent Bacillus anthracis strain 7700 genomic library. Two oligonucleotides derived from the Ba813 sequence were used as primers in polymerase chain reaction tests on genomic DNA from 28 Bacillus anthracis and from 33 heterologous bacteria strains. A specific, 152-bp long DNA fragment was amplified only when Bacillus anthracis DNA was used as the target. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide C1 was covalently linked onto aminated wells of microtiter plates. The detection oligonucleotide D3 was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. Amplification of Ba813 sequence may provide the basis for rapid and reliable assay for the detection and identification of Bacillus anthracis.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  K. Wilson,et al.  Genetic variability of Bacillus anthracis and related species , 1995, Journal of clinical microbiology.

[3]  J. Guesdon,et al.  Rapid detection of Salmonella subspecies I by PCR combined with non-radioactive hybridisation using covalently immobilised oligonucleotide on a microplate. , 1995, FEMS immunology and medical microbiology.

[4]  C. Carlson,et al.  Genotypic Diversity among Bacillus cereus and Bacillus thuringiensis Strains , 1994, Applied and environmental microbiology.

[5]  F. Priest,et al.  Characterization of Bacillus thuringiensis and related bacteria by ribosomal RNA gene restriction fragment length polymorphisms. , 1994, Microbiology.

[6]  T. Reif,et al.  Identification of capsule-forming Bacillus anthracis spores with the PCR and a novel dual-probe hybridization format , 1994, Applied and environmental microbiology.

[7]  M. Johns,et al.  Improved methods for the detection of Bacillus anthracis spores by the polymerase chain reaction , 1994 .

[8]  J. Guesdon,et al.  PCR product quantification by non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells. , 1993, Molecular and cellular probes.

[9]  T. Ezaki,et al.  Direct detection of Bacillus anthracis DNA in animals by polymerase chain reaction , 1993, Journal of clinical microbiology.

[10]  J N Woody,et al.  Detection of spores of Bacillus anthracis using the polymerase chain reaction. , 1992, The Journal of infectious diseases.

[11]  S R Rasmussen,et al.  Covalent immobilization of DNA onto polystyrene microwells: the molecules are only bound at the 5' end. , 1991, Analytical biochemistry.

[12]  A. Fouet,et al.  A target for carbon source-dependent negative regulation of the citB promoter of Bacillus subtilis , 1990, Journal of bacteriology.

[13]  F. Priest,et al.  A numerical classification of the genus Bacillus. , 1988, Journal of general microbiology.

[14]  C. B. Thorne,et al.  Demonstration of a capsule plasmid in Bacillus anthracis , 1985, Infection and immunity.

[15]  I. Uchida,et al.  Association of the encapsulation of Bacillus anthracis with a 60 megadalton plasmid. , 1985, Journal of general microbiology.

[16]  A. Feinberg,et al.  A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. , 1983, Analytical biochemistry.

[17]  B. Ivins,et al.  Evidence for plasmid-mediated toxin production in Bacillus anthracis , 1983, Infection and immunity.

[18]  B. Hohn,et al.  A small cosmid for efficient cloning of large DNA fragments. , 1980, Gene.

[19]  T. Kaneko,et al.  Deoxyribonucleic Acid Relatedness between Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis , 1978, Microbiology and immunology.

[20]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[21]  B. Hohn,et al.  Packaging recombinant DNA molecules into bacteriophage particles in vitro. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[22]  E. Southern.  Detection of specific sequences among DNA fragments separated by gel electrophoresis. , 1975, Journal of molecular biology.

[23]  U. Schibler,et al.  Changes in size and secondary structure of the ribosomal transcription unit during vertebrate evolution. , 1975, Journal of molecular biology.

[24]  W. Haynes,et al.  The genus Bacillus , 1973 .

[25]  C. J. Duggleby,et al.  Differentiation of Bacillus anthracis from other Bacillus cereus group bacteria with the PCR. , 1994, International journal of systematic bacteriology.

[26]  C. J. Duggleby,et al.  Bacillus anthracis but not always anthrax. , 1992, The Journal of applied bacteriology.