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Appl Environ Microbiol. Sep 1993; 59(9): 3011–3020.
PMCID: PMC182400

Detection of the genes encoding botulinum neurotoxin types A to E by the polymerase chain reaction.


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.

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  • Ashton AC, Crowther JS, Dolly JO. A sensitive and useful radioimmunoassay for neurotoxin and its haemagglutinin complex from Clostridium botulinum. Toxicon. 1985;23(2):235–246. [PubMed]
  • Binz T, Kurazono H, Popoff MR, Eklund MW, Sakaguchi G, Kozaki S, Krieglstein K, Henschen A, Gill DM, Niemann H. Nucleotide sequence of the gene encoding Clostridium botulinum neurotoxin type D. Nucleic Acids Res. 1990 Sep 25;18(18):5556–5556. [PMC free article] [PubMed]
  • Binz T, Kurazono H, Wille M, Frevert J, Wernars K, Niemann H. The complete sequence of botulinum neurotoxin type A and comparison with other clostridial neurotoxins. J Biol Chem. 1990 Jun 5;265(16):9153–9158. [PubMed]
  • Durban E, Durban EM, Grecz N. Production of spore spheroplasts of Clostridium botulinum and DNA extraction for density gradient centrifugation. Can J Microbiol. 1974 Mar;20(3):353–358. [PubMed]
  • Fujii N, Kimura K, Murakami T, Indoh T, Yashiki T, Tsuzuki K, Yokosawa N, Oguma K. The nucleotide and deduced amino acid sequences of EcoRI fragment containing the 5'-terminal region of Clostridium botulinum type E toxin gene cloned from Mashike, Iwanai and Otaru strains. Microbiol Immunol. 1990;34(12):1041–1047. [PubMed]
  • Gibson AM, Modi NK, Roberts TA, Shone CC, Hambleton P, Melling J. Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system. J Appl Bacteriol. 1987 Sep;63(3):217–226. [PubMed]
  • Giménez DF, Ciccarelli AS. Another type of Clostridium botulinum. Zentralbl Bakteriol Orig. 1970;215(2):221–224. [PubMed]
  • Hauser D, Eklund MW, Kurazono H, Binz T, Niemann H, Gill DM, Boquet P, Popoff MR. Nucleotide sequence of Clostridium botulinum C1 neurotoxin. Nucleic Acids Res. 1990 Aug 25;18(16):4924–4924. [PMC free article] [PubMed]
  • Johnson JL, Francis BS. Taxonomy of the Clostridia: ribosomal ribonucleic acid homologies among the species. J Gen Microbiol. 1975 Jun;88(2):229–244. [PubMed]
  • Lampel KA, Jagow JA, Trucksess M, Hill WE. Polymerase chain reaction for detection of invasive Shigella flexneri in food. Appl Environ Microbiol. 1990 Jun;56(6):1536–1540. [PMC free article] [PubMed]
  • Lee WH, Riemann H. Correlation of toxic and non-toxic strains of Clostridium botulinum by DNA composition and homology. J Gen Microbiol. 1970 Jan;60(1):117–123. [PubMed]
  • Lo YM, Mehal WZ, Fleming KA. Rapid production of vector-free biotinylated probes using the polymerase chain reaction. Nucleic Acids Res. 1988 Sep 12;16(17):8719–8719. [PMC free article] [PubMed]
  • Miller CA, Anderson AW. Rapid detection and quantitative estimation of type A botulinum toxin by electroimmunodiffusion. Infect Immun. 1971 Aug;4(2):126–129. [PMC free article] [PubMed]
  • Samadpour M, Liston J, Ongerth JE, Tarr PI. Evaluation of DNA probes for detection of Shiga-like-toxin-producing Escherichia coli in food and calf fecal samples. Appl Environ Microbiol. 1990 May;56(5):1212–1215. [PMC free article] [PubMed]
  • Schantz EJ, Johnson EA. Properties and use of botulinum toxin and other microbial neurotoxins in medicine. Microbiol Rev. 1992 Mar;56(1):80–99. [PMC free article] [PubMed]
  • Simpson LL. Molecular pharmacology of botulinum toxin and tetanus toxin. Annu Rev Pharmacol Toxicol. 1986;26:427–453. [PubMed]
  • Sonnabend O, Sonnabend W, Heinzle R, Sigrist T, Dirnhofer R, Krech U. Isolation of Clostridium botulinum type G and identification of type G botulinal toxin in humans: report of five sudden unexpected deaths. J Infect Dis. 1981 Jan;143(1):22–27. [PubMed]
  • Strom MS, Eklund MW, Poysky FT. Plasmids in Clostridium botulinum and related Clostridium species. Appl Environ Microbiol. 1984 Nov;48(5):956–963. [PMC free article] [PubMed]
  • Szabo EA, Pemberton JM, Desmarchelier PM. Specific detection of Clostridium botulinum type B by using the polymerase chain reaction. Appl Environ Microbiol. 1992 Jan;58(1):418–420. [PMC free article] [PubMed]
  • Thomas RJ. Detection of Clostridium botulinum types C and D toxin by ELISA. Aust Vet J. 1991 Mar;68(3):111–113. [PubMed]
  • Thompson DE, Brehm JK, Oultram JD, Swinfield TJ, Shone CC, Atkinson T, Melling J, Minton NP. The complete amino acid sequence of the Clostridium botulinum type A neurotoxin, deduced by nucleotide sequence analysis of the encoding gene. Eur J Biochem. 1990 Apr 20;189(1):73–81. [PubMed]
  • Vary PH, Andersen PR, Green E, Hermon-Taylor J, McFadden JJ. Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne's disease. J Clin Microbiol. 1990 May;28(5):933–937. [PMC free article] [PubMed]
  • Whelan SM, Elmore MJ, Bodsworth NJ, Brehm JK, Atkinson T, Minton NP. Molecular cloning of the Clostridium botulinum structural gene encoding the type B neurotoxin and determination of its entire nucleotide sequence. Appl Environ Microbiol. 1992 Aug;58(8):2345–2354. [PMC free article] [PubMed]
  • Wu JI, Riemann H, Lee WH. Thermal stability of the deoxyribonucleic acid hybrids between the proteolytic strains of Clostridium botulinum and Clostridium sporogenes. Can J Microbiol. 1972 Jan;18(1):97–99. [PubMed]

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