Detection of the Bacillus anthracis gyrA Gene by Using a Minor Groove Binder Probe

ABSTRACT Identification of chromosomal markers for rapid detection of Bacillus anthracis is difficult because significant chromosomal homology exists among B. anthracis, Bacillus cereus, and Bacillus thuringiensis. We evaluated the bacterial gyrA gene as a potential chromosomal marker for B. anthracis. A real-time PCR assay was developed for the detection of B. anthracis. After analysis of the unique nucleotide sequence of the B. anthracis gyrA gene, a fluorescent 3′ minor groove binding probe was tested with 171 organisms from 29 genera of bacteria, including 102 Bacillus strains. The assay was found to be specific for all 43 strains of B. anthracis tested. In addition, a test panel of 105 samples was analyzed to evaluate the potential diagnostic capability of the assay. The assay showed 100% specificity, demonstrating the usefulness of the gyrA gene as a specific chromosomal marker for B. anthracis.

[1]  G. Patra,et al.  Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA. , 1996, FEMS microbiology letters.

[2]  E. Lukhtanov,et al.  3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. , 2000, Nucleic acids research.

[3]  L. Price,et al.  Multiple-Locus Variable-Number Tandem Repeat Analysis Reveals Genetic Relationships within Bacillus anthracis , 2000, Journal of bacteriology.

[4]  The chromosome map of Bacillus thuringiensis subsp. canadensis HD224 is highly similar to that of the Bacillus cereus type strain ATCC 14579. , 1996, FEMS microbiology letters.

[5]  J. Valéro,et al.  Comparative analysis of the 16S to 23S ribosomal intergenic spacer sequences of Bacillus thuringiensis strains and subspecies and of closely related species , 1995, Applied and environmental microbiology.

[6]  D. Linton,et al.  PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples , 1997, Journal of clinical microbiology.

[7]  K. Derbyshire,et al.  Unconventional conjugal DNA transfer in mycobacteria , 2003, Nature Genetics.

[8]  D. Raoult,et al.  rpoB Gene Analysis as a Novel Strategy for Identification of Spirochetes from the Genera Borrelia,Treponema, and Leptospira , 2000, Journal of Clinical Microbiology.

[9]  Jochen Hampe,et al.  An integrated system for high throughput TaqManTM based SNP genotyping , 2001, Bioinform..

[10]  J. M. González,et al.  Transfer of Bacillus thuringiensis plasmids coding for delta-endotoxin among strains of B. thuringiensis and B. cereus. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[11]  N. Saunders,et al.  Identification of species of the genus Legionella using a cloned rRNA gene from Legionella pneumophila. , 1988, Journal of general microbiology.

[12]  C. Carlson,et al.  A complete physical map of a Bacillus thuringiensis chromosome , 1993, Journal of bacteriology.

[13]  W. G. McDonald.  Perforation and hemorrhage after gastrointestinal mucosal biopsy in a child. , 1966, Gastroenterology.

[14]  D. Swinkels,et al.  Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes) , 2002, Human mutation.

[15]  C. B. Thorne,et al.  Involvement of Tn4430 in transfer of Bacillus anthracis plasmids mediated by Bacillus thuringiensis plasmid pXO12 , 1989, Journal of bacteriology.

[16]  K. Livak,et al.  Allelic discrimination using fluorogenic probes and the 5' nuclease assay. , 1999, Genetic analysis : biomolecular engineering.

[17]  W. Goebel,et al.  Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis , 1978, Journal of bacteriology.

[18]  C. B. Thorne,et al.  Identification of self-transmissible plasmids in four Bacillus thuringiensis subspecies , 1987, Journal of bacteriology.

[19]  E. Bode,et al.  Use of Denaturing High-Performance Liquid Chromatography To Identify Bacillus anthracis by Analysis of the 16S-23S rRNA Interspacer Region and gyrA Gene , 2003, Journal of Clinical Microbiology.

[20]  T. Trotta,et al.  Detection of anthrax vaccine virulence factors by polymerase chain reaction. , 2001, Vaccine.

[21]  M. Collins,et al.  Comparative analysis of 23S ribosomal RNA gene sequences of Bacillus anthracis and emetic Bacillus cereus determined by PCR-direct sequencing. , 1992, FEMS microbiology letters.

[22]  J. Melling,et al.  Identification of Bacillus anthracis by API tests. , 1985, Journal of medical microbiology.

[23]  James Pannucci,et al.  Bacillus anthracis pXO1 Plasmid Sequence Conservation among Closely Related Bacterial Species , 2002, Journal of bacteriology.

[24]  Y. Mo,et al.  Characterization of the 23S and 5S rRNA genes of Coxiella burnetii and identification of an intervening sequence within the 23S rRNA gene , 1995, Journal of bacteriology.

[25]  T. Quinn,et al.  Identification of Chlamydia pneumoniae by DNA amplification of the 16S rRNA gene , 1992, Journal of clinical microbiology.

[26]  G. Brightwell,et al.  Fluorescent detection techniques for real‐time multiplex strand specific detection of Bacillus anthracis using rapid PCR , 1999, Journal of applied microbiology.

[27]  H. Cheun,et al.  Detection of anthrax spores from the air by real‐time PCR , 2001, Letters in applied microbiology.

[28]  H. Leclerc,et al.  Biological specificity of bottled natural mineral waters: characterization by ribosomal ribonucleic acid gene restriction patterns. , 1993, The Journal of applied bacteriology.

[29]  F. Dewhirst,et al.  Two types of 16S rRNA gene are found in Campylobacter helveticus: analysis, applications and characterization of the intervening sequence found in some strains. , 1994, Microbiology.

[30]  J. Eshleman,et al.  Use of single nucleotide polymorphisms (SNP) and real-time polymerase chain reaction for bone marrow engraftment analysis. , 2000, The Journal of molecular diagnostics : JMD.

[31]  N. Mann,et al.  Characterization and sequence analysis of a small plasmid from Bacillus thuringiensis var. kurstaki strain HD1-DIPEL. , 1991, Plasmid.

[32]  L. Hall,et al.  Heterogeneity among 16S-23S rRNA intergenic spacers of species within the 'Streptococcus milleri group'. , 1995, Microbiology.

[33]  D. Raoult,et al.  rpoB Gene Sequence-Based Identification of Staphylococcus Species , 2002, Journal of Clinical Microbiology.

[34]  G. Patra,et al.  Utilization of the rpoB Gene as a Specific Chromosomal Marker for Real-Time PCR Detection of Bacillus anthracis , 2001, Applied and Environmental Microbiology.

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

[36]  R. Cox,et al.  The 16S ribosomal RNA of Mycobacterium leprae contains a unique sequence which can be used for identification by the polymerase chain reaction. , 1991, Journal of medical microbiology.

[37]  Thomas F. Smith,et al.  Detection of Bacillus anthracis DNA by LightCycler PCR , 2002, Journal of Clinical Microbiology.

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

[39]  Didier Raoult,et al.  rpoB sequence analysis as a novel basis for bacterial identification , 1997, Molecular microbiology.

[40]  C. Sorlini,et al.  A Randomly Amplified Polymorphic DNA Marker Specific for the Bacillus cereus Group Is Diagnostic forBacillus anthracis , 1999, Applied and Environmental Microbiology.

[41]  G. Patra,et al.  The Ba813 chromosomal DNA sequence effectively traces the whole Bacillus anthracis community , 1999, Journal of applied microbiology.

[42]  R. Kaspar,et al.  Purification and physical analysis of Bacillus anthracis plasmids pXO1 and pXO2. , 1987, Biochemical and biophysical research communications.

[43]  Anne-Brit Kolstø,et al.  Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—One Species on the Basis of Genetic Evidence , 2000, Applied and Environmental Microbiology.

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

[45]  C. B. Thorne,et al.  Mating system for transfer of plasmids among Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis , 1985, Journal of bacteriology.

[46]  C. Carlson,et al.  Physical maps of the genomes of three Bacillus cereus strains , 1992, Journal of bacteriology.

[47]  M. Collins,et al.  Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA. , 1991, International journal of systematic bacteriology.

[48]  P. Glöckner,et al.  A nested PCR method for the detection of Bacillus anthracis in environmental samples collected from former tannery sites. , 1995, Microbiological research.

[49]  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.

[50]  C. Carlson,et al.  A small (2.4 Mb) Bacillus cereus chromosome corresponds to a conserved region of a larger (5.3 Mb) Bacillus cereus chromosome , 1994, Molecular microbiology.

[51]  D. Raoult,et al.  rpoB Sequence Analysis of CulturedTropheryma whippelii , 2001, Journal of Clinical Microbiology.

[52]  C. B. Thorne,et al.  Interspecies transduction of plasmids among Bacillus anthracis, B. cereus, and B. thuringiensis , 1984, Journal of bacteriology.

[53]  M. Gellert,et al.  The DNA dependence of the ATPase activity of DNA gyrase. , 1984, The Journal of biological chemistry.