Detection of the
            <i>Bacillus anthracis gyrA</i>
            Gene by Using a Minor Groove Binder Probe

Hurtle, William; Bode, Elizabeth; Kulesh, David A.; Kaplan, Ronald M.; Garrison, Jeff; Bridge, Deanna L.; House, Michelle; Frye, Melissa S.; Loveless, Bonnie M.; Norwood, David

doi:10.1128/jcm.42.1.179-185.2004

Cited by 107 publications

(70 citation statements)

References 58 publications

(42 reference statements)

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“…One possibility for this failure is that variants other than the 49 strains of B. cereus and B. thuringiensis (testing reported in the original publication) were encountered (11 ). Another explanation is that the large amount of starting material present in boil preps may cause nonspecific amplification to occur, an explanation we consider unlikely, because gel images of the PCR product show that the samples that caused the most false positives (circled in Fig.…”

Section: Discussionmentioning

confidence: 89%

“…Accordingly, a chromosomal assay may augment the ability to distinguish B. anthracis from other environmental Bacillus isolates, but unfortunately there are few specific chromosomal targets to differentiate B. anthracis from other Bacillus species. Until now, the gyrA sequence in B. anthracis has appeared to be unique; however, the singlebase mutation present in near neighbors has proved difficult to reliably reject even after 6 TaqMan-MGB probe designs (11 ). Although TaqMan-MGB has been used with greater success in the gyrA assay at an annealing temperature of 67°C, this temperature departs from our standard protocol, precluding the option of multiplexing this chromosomal assay with virulence plasmid genes, and still reported 7 false positives out of 29 samples.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Tentacle Probes™: Differentiation of Difficult Single-Nucleotide Polymorphisms and Deletions by Presence or Absence of a Signal in Real-Time PCR

et al. 2007

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Section: Discussionmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Tentacle Probes™: Differentiation of Difficult Single-Nucleotide Polymorphisms and Deletions by Presence or Absence of a Signal in Real-Time PCR

et al. 2007

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“…[13][14][15] Finally, a few single nucleotide polymorphisms (SNP) have also been considered for PCR markers. Target genes include rpoB, 24,[48][49][50][51] gyrA, 25,52,53 gyrB, 54,55 plc, 20,23,53,56 purA, 57 and the 16S-23S rDNA internal spacer sequences. [58][59][60] But, so far, only the nonsense mutation in the global regulator PlcR, which controls the transcription of secreted virulence factors in B. cereus and B. thuringiensis, have proved to be truly unique to B. anthracis strains.…”

Section: Literature Survey Of Pcr-based Detection Methodsmentioning

confidence: 99%

“…16,20,59 False-positive signals have sometimes been recorded with closely related strains of the B. cereus group using the other published SNPs. 24,49,52,59,[61][62][63] In silico analysis About a hundred sequences corresponding to all primers and probes currently published were compiled and compared using the primer alignment function of the Gegenees software (www. gegenees.org).…”

Section: Literature Survey Of Pcr-based Detection Methodsmentioning

confidence: 99%

In silico and in vitro evaluation of PCR-based assays for the detection ofBacillus anthracischromosomal signature sequences

et al. 2013

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“…Recent work by repetitive PCR has shown that the previously listed species of Bacillus, as well as Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis, do have some genetic differences (3). Real-time PCR assays based on the chromosomal rpoB and gyrA genes of B. anthracis have been developed (5,13,25). However, these assays are based on single-nucleotide differences between B. anthracis and other Bacillus species.…”

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confidence: 99%

Real-Time PCR Assay for a Unique Chromosomal Sequence of Bacillus anthracis

2004

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Real-time PCR has become an important method for the rapid identification of Bacillus anthracis since the 2001 anthrax mailings. Most real-time PCR assays for B. anthracis have been developed to detect virulence genes located on the pXO1 and pXO2 plasmids. In contrast, only two published chromosomal targets exist, the rpoB gene and the gyrA gene. In the present study, subtraction-hybridization with a plasmid-cured B. anthracis tester strain and a Bacillus cereus driver was used to find a unique chromosomal sequence. By targeting this region, a real-time assay was developed with the Ruggedized Advanced Pathogen Identification Device. Further testing has revealed that the assay has 100% sensitivity and 100% specificity, with a limit of detection of 50 fg of DNA. The results of a search for sequences with homology with the BLAST program demonstrated significant alignment to the recently published B. anthracis Ames strain, while an inquiry for protein sequence similarities indicated homology with an abhydrolase from B. anthracis strain A2012. The importance of this chromosomal assay will be to verify the presence of B. anthracis independently of plasmid occurrence.Bacillus anthracis is a spore-forming gram-positive bacterium well known for its recent use as a bioterrorist agent. Identification of B. anthracis can be done clinically by Gram stain, colony morphology, and various biochemical tests (19). However, these methods are time-consuming, and more rapid tests, such as PCR, have been used to detect B. anthracis in clinical samples (20). Real-time PCR is preferred over conventional PCR methods for the identification of organisms because it is fast, is less labor-intensive, and adds the specificity of a probe. While real-time PCR assays have been used to identify B. anthracis on the basis of the virulence genes associated with the toxin-encoding plasmid (pXO1) and the capsule-encoding plasmid (pXO2) (11,20,22), a reliable chromosomal assay has not been developed. Chromosomal assays can be valuable tools when they are used in conjunction with virulence gene assays because they provide information on the genetic contexts of the pXO1 and pXO2 plasmids. While the chromosomal assays may not prove useful as initial screening assays, they can certainly have a significant role in confirmatory testing as part of an integrated diagnostic approach.Past attempts to develop a chromosomal real-time PCR assay have failed due to the close genetic relationship of Bacillus species. B. anthracis, Bacillus cereus, and Bacillus thuringiensis have very little variability and are genetically indistinguishable by multilocus enzyme electrophoresis (10). Recent work by repetitive PCR has shown that the previously listed species of Bacillus, as well as Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis, do have some genetic differences (3). Real-time PCR assays based on the chromosomal rpoB and gyrA genes of B. anthracis have been developed (5, 13, 25). However, these assays are based on single-nucleotide differences...

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

Cited by 107 publications

References 58 publications

Tentacle Probes™: Differentiation of Difficult Single-Nucleotide Polymorphisms and Deletions by Presence or Absence of a Signal in Real-Time PCR

Tentacle Probes™: Differentiation of Difficult Single-Nucleotide Polymorphisms and Deletions by Presence or Absence of a Signal in Real-Time PCR

In silico and in vitro evaluation of PCR-based assays for the detection ofBacillus anthracischromosomal signature sequences

Real-Time PCR Assay for a Unique Chromosomal Sequence of Bacillus anthracis

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