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2010-04-30Zeitschriftenartikel DOI: 10.1128/AEM.02490-09
Pentaplexed quantitative real-time PCR assay for the simultaneous detection and quantification of botulinum neurotoxin-producing clostridia in food and clinical samples
dc.contributor.authorKirchner, Sebastian
dc.contributor.authorKrämer, Melanie
dc.contributor.authorSchulze, Martin
dc.contributor.authorPauly, Diana
dc.contributor.authorJacob, Daniela
dc.contributor.authorGessler, Frank
dc.contributor.authorNitsche, Andreas
dc.contributor.authorDorner, Brigitte
dc.contributor.authorDorner, Martin
dc.date.accessioned2018-05-07T14:54:17Z
dc.date.available2018-05-07T14:54:17Z
dc.date.created2011-10-18
dc.date.issued2010-04-30none
dc.identifier.otherhttp://edoc.rki.de/oa/articles/reiOuWocDXtB6/PDF/29G1Vh06i9U6w.pdf
dc.identifier.urihttp://edoc.rki.de/176904/972
dc.description.abstractBotulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 10(3) and 10(5) CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F.eng
dc.language.isoeng
dc.publisherRobert Koch-Institut, Biologische Sicherheit
dc.subjectAnimalseng
dc.subjectHumanseng
dc.subjectSerotypingeng
dc.subjectSensitivity and Specificityeng
dc.subjectFood Microbiologyeng
dc.subjectInfanteng
dc.subjectCattleeng
dc.subjectPolymerase Chain Reaction/methodseng
dc.subjectFeces/microbiologyeng
dc.subjectBotulinum Toxins/analysiseng
dc.subjectFood Contamination/analysiseng
dc.subjectBird Diseases/diagnosiseng
dc.subjectBird Diseases/microbiologyeng
dc.subjectBotulism/diagnosiseng
dc.subjectBotulism/microbiologyeng
dc.subjectBotulism/veterinaryeng
dc.subjectCattle Diseases/diagnosiseng
dc.subjectCattle Diseases/microbiologyeng
dc.subjectClostridium botulinum*/classificationeng
dc.subjectClostridium botulinum*/geneticseng
dc.subjectClostridium botulinum*/isolation & purificationeng
dc.subjectClostridium botulinum*/pathogenicityeng
dc.subjectDucks/microbiologyeng
dc.subjectNeurotoxins/analysiseng
dc.subjectTaq Polymeraseeng
dc.subject.ddc610 Medizin
dc.titlePentaplexed quantitative real-time PCR assay for the simultaneous detection and quantification of botulinum neurotoxin-producing clostridia in food and clinical samples
dc.typeperiodicalPart
dc.identifier.urnurn:nbn:de:0257-10015743
dc.identifier.doi10.1128/AEM.02490-09
dc.identifier.doihttp://dx.doi.org/10.25646/897
local.edoc.container-titleApplied and Environmental Microbiology
local.edoc.container-textKirchner, S., Melanie Krämer, K., Schulze, M., Pauly, D., Jacob, D., Gessler, F., Nitsche, A., Dorner, B.G., Dorner, M.B. Pentaplexed quantitative real-time PCR assay for the simultaneous detection and quantification of botulinum neurotoxin-producing clostridia in food and clinical samples (2010) Applied and Environmental Microbiology, 76 (13), pp. 4387-4395.
local.edoc.fp-subtypeArtikel
local.edoc.type-nameZeitschriftenartikel
local.edoc.container-typeperiodical
local.edoc.container-type-nameZeitschrift
local.edoc.container-urlhttp://aem.asm.org/cgi/content/full/76/13/4387
local.edoc.container-publisher-nameAmerican Society for Microbiology
local.edoc.container-volume76
local.edoc.container-issue13
local.edoc.container-year2010

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