title: Pentaplexed quantitative real-time PCR assay for the simultaneous detection and quantification of botulinum neurotoxin-producing clostridia in food and clinical samples.
abstract: Botulinum 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.
authors.affiliation: Center for Biological Safety, Microbial Toxins (ZBS3), Robert Koch-Institute, Nordufer 20, 13353 Berlin, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kirchner
authors.forename: K Melanie
authors.initials: KM
authors.lastname: Krämer
authors.forename: Martin
authors.initials: M
authors.lastname: Schulze
authors.forename: Diana
authors.initials: D
authors.lastname: Pauly
authors.forename: Daniela
authors.initials: D
authors.lastname: Jacob
authors.forename: Frank
authors.initials: F
authors.lastname: Gessler
authors.forename: Andreas
authors.initials: A
authors.lastname: Nitsche
authors.forename: Brigitte G
authors.initials: BG
authors.lastname: Dorner
authors.forename: Martin B
authors.initials: MB
authors.lastname: Dorner
mesh_terms: D000818:Animals; D001715:Bird Diseases / Q000175:diagnosis / Q000382:microbiology; D001905:Botulinum Toxins / Q000032:analysis*; D001906:Botulism / Q000175:diagnosis* / Q000382:microbiology / Q000662:veterinary; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000382:microbiology; D003014:Clostridium botulinum / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D004372:Ducks / Q000382:microbiology; D005243:Feces / Q000382:microbiology; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D006801:Humans; D007223:Infant; D009498:Neurotoxins / Q000032:analysis*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D012703:Serotyping; D019914:Taq Polymerase
chemical_list: D009498:Neurotoxins; D019914:Taq Polymerase; D001905:Botulinum Toxins