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Identification and typing of F rancisella tularensis with a highly automated genotyping assay
Author(s) -
Duncan D.D.,
Vogler A.J.,
Wolcott M.J.,
Li F.,
Sarovich D.S.,
Birdsell D.N.,
Watson L.M.,
Hall T.A.,
Sampath R.,
Housley R.,
Blyn L.B.,
Hofstadler S.A.,
Ecker D.J.,
Keim P.,
Wagner D.M.,
Eshoo M.W.
Publication year - 2013
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/lam.12022
Subject(s) - francisella tularensis , typing , genotyping , biology , microbiology and biotechnology , identification (biology) , polymerase chain reaction , genotype , virology , computational biology , genetics , virulence , gene , botany
A PCR assay was developed to genotypically characterize F rancisella tularensis and F . novicida . An integrated and partially redundant set of markers was selected to provide positive identification of these species, identify subspecies of F . tularensis and genotype 14 variable number tandem repeat ( VNTR ) markers. Assay performance was evaluated with 117 F rancisella samples. Sample DNA was amplified, and the masses of the PCR products were determined with electrospray ionization/time of flight mass spectrometry ( ESI ‐ MS ). The base compositions of the PCR amplicons were derived from these high‐accuracy mass measurements and contrasted with databased information associated with each of the 25 assay markers. Species and subspecies determinations for all samples were fully concordant with results from established typing methods, and VNTR markers provided additional discrimination among samples. Sequence variants were observed with a number of assay markers, but these did not interfere with sample characterization, and served to increase the genetic diversity detected by the assay. Significance and Impact of the Study Molecular characterization of pathogenic subspecies of Francisella tularensis can be obscured by their near neighbours, which can be prevalent in environmental samples. This study describes a multilocus PCR assay for characterizing key species and subspecies of Francisella at multiple levels of resolution, using stable markers defining phylogenetic groups, together with more variable strain‐defining markers. Target loci were selected to form an integrated and semi‐redundant panel enabling robust identification of samples, including unique samples showing novel assay signatures. Analyses of environmental isolates and a diverse panel of curated samples exemplified the applications of this approach in surveillance and outbreak studies.