Real-time PCR for Clostridium botulinum type C neurotoxin (BoNTC) gene, also covering a chimeric C/D sequence—Application on outbreaks of botulism in poultry
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Botulism
Clostridium botulinum
Botulism is a severe neuroparalytic disorder that can be potentially life-threatening. In Barcelona, Spain, no outbreaks had been reported in the past 25 years. However, in September 2011, two outbreaks occurred involving two different families. A rare case of Clostridium baratii which produced a neurotoxin F outbreak was detected in five family members who had shared lunch, and several days before that another family was affected by C. botulinum toxin A which was probably present in homemade pâté.
Botulism
Clostridium botulinum
Neurotoxin
Botulinum neurotoxin
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A polymerase chain reaction (PCR)-based method was established to detect each type of neurotoxin genes of Clostridium botulinum types A to F by employing the oligonucleotide primer sets corresponding to special regions of the light chains of the neurotoxins. In this procedure, the PCR products were easily confirmed by restriction enzyme digestion profiles, and as little as 2.5 pg of template DNAs from toxigenic strains could be detected. The specific PCR products were obtained from toxigenic C. botulinum types A to F, a type E toxin-producing C. butyricum strain, and a type F toxin-producing C. baratii strain, but no PCR product was detected in nontoxigenic strains of C. botulinum and other clostridial species. The neurotoxin genes were also detected in food products of a seasoned dry salmon and a fermented fish (Izushi) which had caused type E outbreaks of botulism. Therefore, it is concluded that this PCR-based detection method can be used for the rapid diagnosis of botulism.
Botulism
Clostridium botulinum
Neurotoxin
Food poisoning
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The polymerase chain reaction (PCR) was used as the basis for the development of highly sensitive and specific diagnostic tests for organisms harboring botulinum neurotoxin type A through E genes. Synthetic DNA primers were selected from nucleic acid sequence data for Clostridium botulinum neurotoxins. Individual components of the PCR for each serotype (serotypes A through E) were adjusted for optimal amplification of the target fragment. Each PCR assay was tested with organisms expressing each of the botulinum neurotoxin types (types A through G), Clostridium tetani, genetically related nontoxigenic organisms, and unrelated strains. Each assay was specific for the intended target. The PCR reliably identified multiple strains having the same neurotoxin type. The sensitivity of the test was determined with different concentrations of genomic DNA from strains producing each toxin type. As little as 10 fg of DNA (approximately three clostridial cells) was detected. C. botulinum neurotoxin types A, B, and E, which are most commonly associated with human botulism, could be amplified from crude DNA extracts, from vegetative cells, and from spore preparations. This suggests that there is great potential for the PCR in the identification and detection of botulinum neurotoxin-producing strains.
Clostridium botulinum
Neurotoxin
Botulism
Botulinum neurotoxin
genomic DNA
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A quantitative real-time PCR (qPCR) for detection of the neurotoxin of the Clostridium botulinum type C (BoNTC) encoding gene has been compared with a nested PCR (nPCR) and a conventional PCR (cPCR) using 2 toxigenic C. botulinum C1 reference strains and samples from bird tissues (n = 30) and sediments (n = 30) from wetlands where botulism outbreaks have been reported. A cPCR based on 16S ribosomal RNA sequences from 60 strains of Clostridium species was also developed to detect the genomic DNA of C. botulinum C in order to evaluate the presence of nontoxigenic strains. Quantitative PCR showed a similar sensitivity to nPCR (<0.5 pg of DNA), and both were more sensitive than the cPCR when using the C. botulinum reference strains. Quantitative PCR and nPCR revealed an equal number of positives in uncultured samples of sediments (3%) and bird tissues (40%), but these values tended to be higher after culture enrichment with the qPCR assay (10% and 80%, respectively). Associations between the presences of toxigenic C. botulinum C in the environment and in birds within the ecological conditions in wetlands could be studied further using the culture enrichment and qPCR techniques shown in the current study.
Clostridium botulinum
Botulism
genomic DNA
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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.
Clostridium botulinum
Botulism
TaqMan
Multiplex
Botulinum neurotoxin
Clostridia
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Real-time PCR assays for detection of Clostridium botulinum neurotoxin (BoNT) gene fragments specific to BoNTA, B, and E were developed as alternatives to the mouse bioassay. The expected specificities of the PCR assays were demonstrated by in silico analysis as well as empirical testing of target DNA extracted from 83 pure cultures of C. botulinum, and 44 bacteria from other species. The sensitivities of the assays were found to be equivalent to 16, 10, and 141 genomes for BoNT A, B, and E, respectively. The assays were shown to be applicable to both purified DNA, as well as crude DNA extracted from cultures and enrichment broths. The assays were evaluated using DNA extracted directly from clinical and food specimens as well as from inoculated broths using material collected from seven confirmed and one suspected case of botulism. The appropriate BoNT genes were detected in material from seven of the eight cases of botulism and provided a supportive diagnosis faster than the conventional bioassay. These assays have already proven useful for pubic health microbiological investigation of suspected cases of human botulism by substantially improving the diagnostic process.
Botulism
Clostridium botulinum
Neurotoxin
Botulinum neurotoxin
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Clostridium botulinum
Botulism
Neurotoxin
Assay sensitivity
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Botulism
Clostridium botulinum
Neurotoxin
Botulinum neurotoxin
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An animal model of wound botulism was developed in mice using an inoculum of Clostridium botulinum type A spores. The number of C. botulinum in infected wounds was quantitated by culturing on egg yolk agar, and the level of C. botulinum toxin in infected wound tissue was measured by a bioassay in mice and by an enzyme-linked immunosorbent assay. All infected mice receiving no further treatment developed neuroparalytic symptoms consistent with botulism after an incubation period of ca. 48 h, and all of these animals died. Serotherapy with C. botulinum type A antitoxin initiated 24 h postchallenge reduced the mortality rate to 5%. Treatment with metronidazole 2 to 24 h postchallenge resulted in recovery rates of 40 to 91%.
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Clostridium botulinum
Antitoxin
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An atypical toxin variant of Clostridium botulinum (strain 657) was isolated from the feces of a 6-week-old female infant whose symptoms and clinical history were consistent with infant botulism. Toxin detected in the feces and the toxin produced by isolates from the feces and from two rectal swabs could be neutralized by type B botulinal antitoxin only at very high ratios of of antitoxin to toxin in the neutralization mixture. One international unit of type B antitoxin neutralized only about 10 lethal doses of 657 toxin as compared with approximately 10,000 lethal doses of conventional type B toxin from the Beans strain. Antitoxin prepared against 657 toxin was 10 times more effective against the conventional toxin than against the homologous toxin. Toxoid-antitoxin-binding studies indicate that both 657 toxin and type B toxin are heterogeneous and that both toxins may contain the same molecular variants, but that the proportions of the variants are different in each.
Antitoxin
Botulism
Clostridium botulinum
Toxoid
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