Detection of
            <i>Bacillus anthracis</i>
            DNA by LightCycler PCR

Bell, Constance A.; Uhl, James R.; Hadfield, Ted L.; David, J. C.; Meyer, Richard F.; Smith, Thomas F.; Cockerill, Franklin R.

doi:10.1128/jcm.40.8.2897-2902.2002

Cited by 132 publications

(79 citation statements)

References 23 publications

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“…Polymerase chain reactions (PCR) or multiplication of the microorganism through culture growth are used to generate the millions of copies that are required to accurately sequence the nucleic acid. Although B. anthracis identification by PCR has been demonstrated by sequencing the capsular protein B gene in under 1 h, 3 the CDC required several days to identify the contents of the letter addressed to Senator Tom Daschle as B. anthracis, and longer still to verify that it was the deadly Ames strain. 1 Immunoassay methods are also being developed that use competitive binding of the bioagent (as an antigen) and its labeled conjugate for a limited number of antibodies.…”

Section: Introductionmentioning

confidence: 99%

Detecting Bacillus cereus spores on a mail sorting system using Raman spectroscopy

Farquharson

Grigely

Khitrov

et al. 2004

J Raman Spectroscopy

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The ability of Raman spectroscopy to detect anthrax-causing spores as they pass through a mail sorting system was investigated. A pump was connected to an existing vacuum manifold on a commercial sorter, and a filter designed to capture 0.5-3 µm particles was placed in-line. A standard business letter containing 0.23 g of Bacillus cereus spores, a Bacillus anthracis surrogate, was placed in a stack of 20 letters and passed through the system. Raman spectra of the filter positively identified the captured material as bacterial spores by the dominant calcium dipicolinate Raman spectral bands associated with the spore core. A limit of detection, using 400 mW of 785 nm laser excitation for a 1-s acquisition, is estimated at 4.5 mg. The ability of a Raman spectroscopy based system to detect and prevent the distribution of a letter containing gram levels of anthrax spores is discussed.

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Section: Introductionmentioning

confidence: 99%

Detecting Bacillus cereus spores on a mail sorting system using Raman spectroscopy

Farquharson

Grigely

Khitrov

et al. 2004

J Raman Spectroscopy

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“…Anthrax detection can be performed by amplifying the virulence plasmids (23)(24)(25), but as with Y. pestis, there is concern over genetic modification of near neighbors and false negatives from B. anthracis without one or more of the plasmids. Moreover, B. cereus and Bacillus thuringiensis isolates have been found that harbor 1 or both B. anthracis plasmids with and without anthrax toxin genes (26 -31 ).…”

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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“…Immunoassays, which rely on the interaction between B. anthracis spore surface antigens and their specific antibodies, can detect 10 5 spores in approximately 12 h. However, in the case of immunoassays, specific antibodies and additional molecular fluorophores and/or enzymatic reactions must be employed for the desired agents to be detected, and mobile-phase conditions must also be adjusted depending on their capture, elution, and separation properties. In addition, although this direct spore detection system is relatively fast, the lack of accuracy and limited sensitivity of current antibody-based detection methods result in unacceptably high levels of both false-positive and false-negative responses [12][13][14]. Therefore, the development of a more robust detection system is required.…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, label-free methods implemented for the specific detection of DNA strands were developed [10,11]. Among the most important biological methods for the detection of anthrax, Polymerase Chain Reaction (PCR) [5][6][7] and immunoassay [12][13][14] have been used as representatives. PCR, a primer-mediated enzymatic DNA amplification method, requires expensive reagents and takes 3-4 h or longer several hours to obtain the result.…”

Section: Introductionmentioning

confidence: 99%

Development of a Separation and Detection System for Bacillus anthracis Spores Based on Peptide Conjugates Identified from Peptide Library

Park¹

2015

Biosens J

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A simple, specific and rapid identification system against Bacillus anthracis spores was developed using specific capture peptides conjugated with a bead-based biosensor, which was tightly and specifically bound onto the spore surface of B. anthracis. We successfully detected and separated B. anthracis spores from the spores of B. thuringiensis and B. cereus using this peptide-magnetic bead conjugates by fluorescence and anthrax-specific analyses. For more convenient mediation of high-throughput detection against B. anthracis spores, streptavidin-biotin interactions of spore-peptide and spore-peptide-magnetic bead conjugates were performed, and we demonstrated the separation and detection of B. anthracis spores using this method. In the presence of mixed condition of several types of Bacillus spores, the B. anthracis spores were easily separated using the oligopeptide-conjugated magnetic beads, thus allowing the clear detection of B. anthracis spores from B. cereus, B. subtilis, and B. thuringiensis spores. This oligopeptidebased strategy was rapidly and unambiguously identified as little as one viable B. anthracis spore in less than 1 h with a simple binding assay format. When assessed for its effectiveness for specifically and selectively detection of environmental spores phylogenetically similar to B. anthracis spores, such as B. thuringiensis and B. cereus spores, the system was free of false-positive signals. In this study, we developed a high-throughput detection system for B. anthracis spores with oligopeptides using fluorescence, magnetic beads, and positional scanning-synthetic peptide combinatorial libraries (PS-SPCLs). Specific capture oligopeptides were developed to specifically bind against the surface of B. anthracis spores. Several peptide ligands were conjugated with Fluorescein Isothiocyanate (FITC) to detect fluorescent signals, and magnetic beads were used to facilitate rapid isolation by magnetic polarity conjugated with streptavidin to bind with biotin-conjugated oligopeptides. The resulting system was capable of the detection of specific spores with high sensitivity in less than 1 h. We also optimized the system to maximize its binding affinity to spores of two B. anthracis strains, namely, B. anthracis ΔSterne Finally, to further improve the performance of the capture peptides, we newly designed their amino acid sequences using PS-SPCLs that can bind B. anthracis spores with greater specificity. The detection system based on these specific capture peptides is a high-throughput fluorescence-based assay that employs quantum dots. These specific binding systems can be employed to rapidly, specifically and selectively detect and separate B. anthracis spores in mixed conditions. Development of a Separation and Detection Materials and Methods MaterialsStreptavidin-conjugated magnetic beads (~ 1-2 μm in diameter) and a portable handheld magnetic separator were purchased from PureBiotech (San Diego, CA). The capture peptides used in this study (Table 1) were synthesized by Peptron (Korea)....

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Detection of Bacillus anthracis DNA by LightCycler PCR

Cited by 132 publications

References 23 publications

Detecting Bacillus cereus spores on a mail sorting system using Raman spectroscopy

Detecting Bacillus cereus spores on a mail sorting system using Raman spectroscopy

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

Development of a Separation and Detection System for Bacillus anthracis Spores Based on Peptide Conjugates Identified from Peptide Library

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