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      Coxiella burnetii Genotyping

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          Abstract

          Multispacer sequence typing is the first reliable method for typing Coxiella burnetii isolates.

          Abstract

          Coxiella burnetii is a strict intracellular bacterium with potential as a bioterrorism agent. To characterize different isolates of C. burnetii at the molecular level, we performed multispacer sequence typing (MST). MST is based on intergenic region sequencing. These regions are potentially variable since they are subject to lower selection pressure than the adjacent genes. We screened 68 spacers in 14 isolates and selected the 10 that exhibited the most variation. These spacers were then tested in 159 additional isolates obtained from different geographic areas or different hosts or were implicated in different manifestations of human disease caused by C. burnetii. The sequence analysis yielded 30 different allelic combinations. Phylogenic analysis showed 3 major clusters. MST allows easy comparison and exchange of results obtained in different laboratories and could be a useful tool for identifying bacterial strains.

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          Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms.

          M. C. J. Maiden, J. Bygraves, E Feil … (1998)
          Traditional and molecular typing schemes for the characterization of pathogenic microorganisms are poorly portable because they index variation that is difficult to compare among laboratories. To overcome these problems, we propose multilocus sequence typing (MLST), which exploits the unambiguous nature and electronic portability of nucleotide sequence data for the characterization of microorganisms. To evaluate MLST, we determined the sequences of approximately 470-bp fragments from 11 housekeeping genes in a reference set of 107 isolates of Neisseria meningitidis from invasive disease and healthy carriers. For each locus, alleles were assigned arbitrary numbers and dendrograms were constructed from the pairwise differences in multilocus allelic profiles by cluster analysis. The strain associations obtained were consistent with clonal groupings previously determined by multilocus enzyme electrophoresis. A subset of six gene fragments was chosen that retained the resolution and congruence achieved by using all 11 loci. Most isolates from hyper-virulent lineages of serogroups A, B, and C meningococci were identical for all loci or differed from the majority type at only a single locus. MLST using six loci therefore reliably identified the major meningococcal lineages associated with invasive disease. MLST can be applied to almost all bacterial species and other haploid organisms, including those that are difficult to cultivate. The overwhelming advantage of MLST over other molecular typing methods is that sequence data are truly portable between laboratories, permitting one expanding global database per species to be placed on a World-Wide Web site, thus enabling exchange of molecular typing data for global epidemiology via the Internet.
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            Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material.

            P Walsh, D Metzger, R Higuchi (1991)
            Procedures utilizing Chelex 100 chelating resin have been developed for extracting DNA from forensic-type samples for use with the PCR. The procedures are simple, rapid, involve no organic solvents and do not require multiple tube transfers for most types of samples. The extraction of DNA from semen and very small bloodstains using Chelex 100 is as efficient or more efficient than using proteinase K and phenol-chloroform extraction. DNA extracted from bloodstains seems less prone to contain PCR inhibitors when prepared by this method. The Chelex method has been used with amplification and typing at the HLA DQ alpha locus to obtain the DQ alpha genotypes of many different types of samples, including whole blood, bloodstains, seminal stains, buccal swabs, hair and post-coital samples. The results of a concordance study are presented in which the DQ alpha genotypes of 84 samples prepared using Chelex or using conventional phenol-chloroform extraction are compared. The genotypes obtained using the two different extraction methods were identical for all samples tested.
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              MEGA2: molecular evolutionary genetics analysis software.

              S. Kumar, K Tamura, I. Jakobsen … (2001)
              We have developed a new software package, Molecular Evolutionary Genetics Analysis version 2 (MEGA2), for exploring and analyzing aligned DNA or protein sequences from an evolutionary perspective. MEGA2 vastly extends the capabilities of MEGA version 1 by: (1) facilitating analyses of large datasets; (2) enabling creation and analyses of groups of sequences; (3) enabling specification of domains and genes; (4) expanding the repertoire of statistical methods for molecular evolutionary studies; and (5) adding new modules for visual representation of input data and output results on the Microsoft Windows platform. http://www.megasoftware.net. s.kumar@asu.edu
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                Author and article information

                Journal
                Journal ID (nlm-ta): Emerg Infect Dis
                Journal ID (iso-abbrev): Emerging Infect. Dis
                Journal ID (publisher-id): EID
                Title: Emerging Infectious Diseases
                Publisher: Centers for Disease Control and Prevention
                ISSN (Print): 1080-6040
                ISSN (Electronic): 1080-6059
                Publication date (Print): August 2005
                Volume: 11
                Issue: 8
                Pages: 1211-1217
                Affiliations
                [* ]Unité des Rickettsies, Marseille, France;
                [† ]Pasteur Institute of Epidemiology and Microbiology, Saint Petersburg, Russia;
                [‡ ]Institute of Virology SAS, Bratislava, Slovak Republic;
                [§ ]University of Giessen, Giessen, Germany;
                []University of Alberta Department of Medicine, Edmonton, Alberta, Canada
                Author notes
                Address for correspondence: Didier Raoult, Unité des Rickettsies, Faculté de Médecine, CNRS UMR 6020, IFR48, 27 Boulevard Jean Moulin, 13385 Marseille, Cedex 05, France; fax: 33-491-32-03 90. email: didier.raoult@ 123456medecine.univ-mrs.fr
                Article
                Publisher ID: 04-1354
                DOI: 10.3201/eid1108.041354
                PMC ID: 3320512
                PubMed ID: 16102309
                SO-VID: 2ca715af-cdab-4d2d-a062-f30a172da420
                History
                Categories
                Subject: Research
                Subject: Research

                ScienceOpen disciplines: Infectious disease & Microbiology
                Keywords: bacterial typing,phylogeny,q fever,keywords: coxiella burnetii,dna sequence analysis
                Data availability:
                ScienceOpen disciplines: Infectious disease & Microbiology
                Keywords: bacterial typing, phylogeny, q fever, keywords: coxiella burnetii, dna sequence analysis

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