Pyrosequencing<i>Bacillus anthracis</i>

Wahab, Tara; Hjalmarsson, Sandra; Wollin, Ralfh; Engstrand, Lars

doi:10.3201/eid1110.041316

Cited by 26 publications

(10 citation statements)

References 22 publications

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“…Pyrosequencing‐based SNP detection in three genes, siaD, porB , and porA , allow a rapid and specific characterization of Neisseria meningitidis strains (Diggle & Clarke, 2004). Pyrosequencing of genomic fragments has been used to genotype Neisseria gonorrhoeae and B. anthracis (Diggle & Clarke, 2004; Unemo et al , 2004; Wahab et al , 2005).…”

Section: Current Methods For Bacterial Strain Typingmentioning

confidence: 99%

Bacterial strain typing in the genomic era

2009

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Bacterial strain typing, or identifying bacteria at the strain level, is particularly important for diagnosis, treatment, and epidemiological surveillance of bacterial infections. This is especially the case for bacteria exhibiting high levels of antibiotic resistance or virulence, and those involved in nosocomial or pandemic infections. Strain typing also has applications in studying bacterial population dynamics. Over the last two decades, molecular methods have progressively replaced phenotypic assays to type bacterial strains. In this article, we review the current bacterial genotyping methods and classify them into three main categories: (1) DNA banding pattern-based methods, which classify bacteria according to the size of fragments generated by amplification and/or enzymatic digestion of genomic DNA, (2) DNA sequencing-based methods, which study the polymorphism of DNA sequences, and (3) DNA hybridization-based methods using nucleotidic probes. We described and compared the applications of genotyping methods to the study of bacterial strain diversity. We also discussed the selection of appropriate genotyping methods and the challenges of bacterial strain typing, described the current trends of genotyping methods, and investigated the progresses allowed by the availability of genomic sequences.

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Section: Current Methods For Bacterial Strain Typingmentioning

confidence: 99%

Bacterial strain typing in the genomic era

2009

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“…Recent comparative full-genome sequencing allowed the discovery of about 3,500 SNPs among eight strains of B. anthracis (15,17; J. Ravel, unpublished data) and represents a valuable resource for developing diagnostic signatures for this pathogen. SNPs are particularly attractive markers for subtyping efforts since they are evolutionarily stable (8,15) and are amenable to high-throughput detection methods, such as realtime PCR, pyrosequencing, and mass spectrometry (1,2,4,10,20,22,23).…”

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confidence: 99%

Strain-Specific Single-Nucleotide Polymorphism Assays for the Bacillus anthracis Ames Strain

et al. 2007

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Highly precise diagnostics and forensic assays can be developed through a combination of evolutionary analysis and the exhaustive examination of genomic sequences. In Bacillus anthracis, whole-genome sequencing efforts revealed ca. 3,500 single-nucleotide polymorphisms (SNPs) among eight different strains and evolutionary analysis provides the identification of canonical SNPs. We have previously shown that SNPs are highly evolutionarily stable, and the clonal nature of B. anthracis makes them ideal signatures for subtyping this pathogen. Here we identified SNPs that define the lineage of B. anthracis that contains the Ames strain, the strain used in the 2001 bioterrorist attacks in the United States. Sequencing and real-time PCR were used to validate these SNPs across B. anthracis strains, including (i) 88 globally and genetically diverse isolates; (ii) isolates that were shown to be genetic relatives of the Ames strain by multiple-locus variable number tandem repeat analysis (MLVA); and (iii) several different lab stocks of the Ames strain, including a clinical isolate from the 2001 letter attack. Six SNPs were found to be highly specific for the Ames strain; four on the chromosome, one on the pX01 plasmid, and one on the pX02 plasmid. All six SNPs differentiated the B. anthracis Ames strain from the 88 unique B. anthracis strains, while five of the six separated Ames from its close genetic relatives. The use of these SNPs coupled with real-time PCR allows specific and sensitive (<100 fg of template DNA) identification of the Ames strain. This evolutionary and genomics-based approach provides an effective means for the discovery of strain-specific SNPs in B. anthracis.The 2001 anthrax letter attack illustrated the "real-world" efficacy of Bacillus anthracis as a bioterror agent. Forensic and epidemiological analysis of clinical samples and weaponized spores from the letter attack included the identification of the B. anthracis strain as Ames (3,8,17). This was initially accomplished using multiple-locus variable number tandem repeat analysis (MLVA) (3,6,8), which was one of the few technical approaches possible for this highly monomorphic pathogen (5). Identifying the strain and establishing its identity in attack locations were important in linking the dispersed anthrax cases and suggesting a possible source for the weaponized material. During the anthrax attack crisis, diagnostic speed, specificity, and sensitivity were often limiting factors, necessitating extraordinary efforts by public health officials and forensic labs (3). Further advancement of molecular diagnostics allows for more-efficient responses in disease outbreaks, whether natural or bioterrorist mediated.Single-nucleotide polymorphisms (SNPs) are increasingly recognized as important markers for detecting and subtyping bacterial pathogens, including B. anthracis (1,2,4,8,16,18,19). Recent comparative full-genome sequencing allowed the discovery of about 3,500 SNPs among eight strains of B. anthracis (15, 17; J. Ravel, unpublished data) and repre...

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“…So far, the method has been used for applications where sequence primers can be designed to produce a read length sufficient for a correct result. Examples include single nucleotide polymorphism (SNP) analysis [25][26][27][28][29], identification and subtyping of bacteria [25], mutation detection in disease genes [30][31], and quantification of and mutation detection in HIV-1 [32][33]. The pyrosequencing technology has been further developed and is today also available as a tool for large-scale sequencing (genome Sequencer FLX, 454 Life Science/Roche).…”

Section: Pyrosequencingmentioning

confidence: 99%

Real-time polymerase chain reaction followed by fast sequencing allows rapid genotyping of microbial pathogens

Wahab

Ankarklev

Lebbad

et al. 2010

Scandinavian Journal of Infectious Diseases

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In this study we describe a novel protocol for rapid molecular analysis of patient samples using a combination of real-time polymerase chain reaction (PCR) and Sanger sequencing. This would normally take 2 working days in the diagnostic laboratory, but using this protocol the process can be completed within 3 h using equipment normally found in the laboratory. The innovative steps in this protocol are the sequencing of the product generated in the diagnostic real-time PCR, addition of a sequencing tail to the PCR primer, which increases the quality of the sequence without loss of sensitivity or specificity, and optimization of the hands-on and instrument steps using modern reagents. The read length of the sequencing step is routinely 250 nucleotides, which is substantially longer than existing rapid sequencing methods, increasing the chances of covering several genetic markers within 1 analysis. As proof of the concept, we used the detection and genotyping of the intestinal parasite Giardia lamblia, but the protocol can be applied to any PCR and sequence-based analysis.

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PyrosequencingBacillus anthracis

Abstract: Pyrosequencing technology was used to rapidly and specifically identify Bacillus anthracis.

Cited by 26 publications

References 22 publications

Bacterial strain typing in the genomic era

Bacterial strain typing in the genomic era

Strain-Specific Single-Nucleotide Polymorphism Assays for the Bacillus anthracis Ames Strain

Real-time polymerase chain reaction followed by fast sequencing allows rapid genotyping of microbial pathogens

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