RAPDおよびPCR-RFLP法によるA型毒素産生ボツリヌス菌の遺伝子型鑑

ボツリヌス菌の分子疫学解析手法として, RAPD法, 神経毒素複合体遺伝子を標的としたPCR法およびPCRRFLP法の有用性について検討した.試験菌株として, 食餌性ボツリヌス症由来株, 日本および米国の乳児ボツリヌス症由来株, 蜂蜜由来株など23株を用いた.RAPD法は, 日本の乳児株および蜂蜜由来804-1H株と他の菌株を明瞭に識別した.一方, 神経毒素 (BoNT/A, BoNT/B), 無毒成分 (nontoxic-nonhemagglutinin (NTNH), hemagglutinin (HA)), およびp47蛋白質の遺伝子解析の結果, 供試A型菌は3つの遺伝子型に分けられた....

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Published in日本食品微生物学会雑誌 Vol. 22; no. 4; pp. 148 - 154
Main Authors 中野, 宏幸, 荻野, 武雄, 石村, 勝之, 萱島, 隆之
Format Journal Article
LanguageEnglish
Published 日本食品微生物学会 2005
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ISSN1340-8267
1882-5982
DOI10.5803/jsfm.22.148

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Abstract ボツリヌス菌の分子疫学解析手法として, RAPD法, 神経毒素複合体遺伝子を標的としたPCR法およびPCRRFLP法の有用性について検討した.試験菌株として, 食餌性ボツリヌス症由来株, 日本および米国の乳児ボツリヌス症由来株, 蜂蜜由来株など23株を用いた.RAPD法は, 日本の乳児株および蜂蜜由来804-1H株と他の菌株を明瞭に識別した.一方, 神経毒素 (BoNT/A, BoNT/B), 無毒成分 (nontoxic-nonhemagglutinin (NTNH), hemagglutinin (HA)), およびp47蛋白質の遺伝子解析の結果, 供試A型菌は3つの遺伝子型に分けられた.このうち, 日本乳児株, 蜂蜜由来804-1H株およびAF84株は共通のBoNT/AおよびNTNH切断パターンを示し, かつHA遺伝子陰性, P47遺伝子陽性でその他の株と異なった.これらの結果から, 今回検討したPCR法に基づいた解析法は, A型毒素産生ボツリヌス菌の簡易で迅速な遺伝子型鑑別法として有用と考えられた.
AbstractList ボツリヌス菌の分子疫学解析手法として, RAPD法, 神経毒素複合体遺伝子を標的としたPCR法およびPCRRFLP法の有用性について検討した.試験菌株として, 食餌性ボツリヌス症由来株, 日本および米国の乳児ボツリヌス症由来株, 蜂蜜由来株など23株を用いた.RAPD法は, 日本の乳児株および蜂蜜由来804-1H株と他の菌株を明瞭に識別した.一方, 神経毒素 (BoNT/A, BoNT/B), 無毒成分 (nontoxic-nonhemagglutinin (NTNH), hemagglutinin (HA)), およびp47蛋白質の遺伝子解析の結果, 供試A型菌は3つの遺伝子型に分けられた.このうち, 日本乳児株, 蜂蜜由来804-1H株およびAF84株は共通のBoNT/AおよびNTNH切断パターンを示し, かつHA遺伝子陰性, P47遺伝子陽性でその他の株と異なった.これらの結果から, 今回検討したPCR法に基づいた解析法は, A型毒素産生ボツリヌス菌の簡易で迅速な遺伝子型鑑別法として有用と考えられた.
Author 荻野, 武雄
石村, 勝之
萱島, 隆之
中野, 宏幸
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  organization: 広島市衛生研究所生物科学部
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  fullname: 石村, 勝之
  organization: 広島市衛生研究所生物科学部
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  fullname: 萱島, 隆之
  organization: 広島市衛生研究所生物科学部
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References 21) Oguma, K., Fujinaga, Y. and Inoue, K.: Structure and function of Clostridium botulinum toxins. Microbiol. Immunol., 39,161-168 (1995).
9) Fujinaga, Y., Takeshi, K., Inoue , K., Fujita, R., Ohyama, T., Moriishi, K. and Oguma, K.: Type A and B neurotoxin genes in a Clostridium botulinum type AB strain. Biochem. Biophys. Res. Commun., 213,737-745 (1995).
4) East, A. K. and Collins, M. D.: Conserved structure of genes encoding components of botulinum neurotoxin complex M and the sequence of the gene coding for the nontoxic component in nonproteolytic Clostridium botulinum type F. Curr. Microbiol., 29, 69-77 (1994).
7) Franciosa, G., Ferreira, J. L. and Hatheway , C. L.: Detection of type A, B, and E botulism neurotoxin genes in Clostridium botulinum and other Clostridium species by PCR: evidence of unexpressed type B toxin genes in type A toxigenic organisms. J. Clin. Microbiol., 32, 1911-1917 (1994).
3) East, A. K., Bhandari, M., Stacey, J. M., Campbell, K. D. and Collins, M. D.: Organization and phylogenetic interrelationships of genes encoding components of the botulinum toxin complex in proteolytic Clostridium botulinum types A, B, and F: evidence of chimeric sequences in the gene encoding the nontoxic nonhemagglutinin component. Int. J. Syst. Bacteriol., 46, 1105-1112 (1996).
15) Kubota, T., Shirakawa, S., Kozaki, S., Isogai, E., Isogai, H., Kimura, K. and Fujii, N.: Mosaic type of the nontoxic- nonhemagglutinin component gene in Clostridium botulinum type A strain isolated from infant botulism in Japan. Biochem. Biophys. Res. Commun., 224,843-848 (1996).
16) Lund, B. M. and Peck, M. W.: Clostridium botulinum, the microbiological safety and quality of food. Lund, B. M., Baird-Parker, A. C., and Gould (eds.), p. 1057-1109, Aspen, Gaithersburg, Md. (2000).
27) Tabita, K., Sakaguchi, S., Kozaki, S. and Sakaguchi, G.: Distinction between Clostridium botulinum type A strains associated with food-borne botulism and those with infant botulism in Japan in intraintestinal toxin production in infant mice and some other properties. FEMS Microbiol. Lett., 79,251-256 (1991).
22) Oguma, K., Inoue, K., Fujinaga, Y., Yokota, K. Watanabe, T., Ohyama, T., Takeshi, K. and Inoue, K.: Structure and function of Clostridium botulinum progenitor toxin. J. Toxicol.-Toxin Rev., 18, 17-34 (1999).
20) Nakano, H., Yoshikuni, Y., Hashimoto, H. and Sakaguchi, G.: Detection of Clostridium botulinum in natural sweetening. Int. J. Food Microbiol., 16,117-121 (1992).
23) Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Hoorn, G. T., Erlich, H. A. and Arnheim, N.: Enzymatic amplification of 13-globin genomic sequences and restriction site analysis for diagnosis of Siekle cell anemia. Science, 230, 1350-1354 (1985).
14) Kozaki, S., Nakaue, S. and Kamata, Y.: Immunological characterization of the neurotoxin produced by Clostridium botulinum type A associated with infant botulism in Japan. Microbiol. Immun., 39,767-774 (1995).
8) Franciosa, G., Floridi, F., Maugliani, A. and Aureli , P.: Differentiation of the gene clusters encoding botulinum neurotoxin type A complexes in Clostridium botulinum type A, Ab, and A (B) strains. Appl. Environ. Microbiol., 70, 7192-7199 (2004).
1) Arnon, S. S.: Infant botulism. Ann. Rev. Med., 31,541-560 (1981).
13) Inoue, K., Fujinaga, Y., Watanabe, T., Ohyama , T., Takeshi, K., Moriishi, K., Nakajima, H., Inoue, K. and Oguma, K.: Molecular composition of Clostridium botulinum type A progenitor toxins. Infect . Immun., 64, 1589-1594 (1996).
5) East, A. K., Stacey, J. M. and Collins , M. D.: Cloning and sequencing of a hemagglutinin component of the botulinum neurotoxin complex encoded by Clostridium botulinum types A and B. System. Appl. Microbiol., 17,306-312 (1994).
11) Hutson, R. A., Collins, M. D., East, A. K. and Thompson, D. E.: Nucleotide sequence of the gene coding for non-proteolytic Clostridium botulinum type B neurotoxin: comparison with other clostridial neurotoxins. Curr. Microbiol., 28,101-110 (1994).
25) Shapiro, R. L., Hatheway, C. and Swerdlow, D. L.: Botulism in the United States: a clinical and epidemiological review. Ann. Intern. Med., 129,221-228 (1998).
26) Tabita, K., Sakaguchi, S., Kozaki, S. and Sakaguchi, G.: Comparative studies on Clostridium botulinum type A strains associated with infant botulism in Japan and in California, USA. Jpn. J. Med. Sci. Biol., 43,219-231 (1990).
2) Cordoba, J. J., Collins, M. D. and East, A. K.: Studies on the genes encoding botulinum neurotoxin type A of Clostridium botulinum from a variety of sources. System. Appl. Microbiol., 18, 13-22 (1995).
10) Henderson, I., Whelan, S. M., Davis, T. O. and Minton, N. P.: Genetic characterization of the botulinum toxin complex of Clostridium botulinum strain NCTC 2916. FEMS Microbiol. Lett., 140,151-158 (1996).
19) Murmar, J.: A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol., 3,208-218 (1961).
28) Takeda, Y., Toukubo, Y., Inoue, K., Ogawa, H., Okada, S. and Sato, T.: Infant botulism by Clostridium botulinum possessing the type A and B botulism neurotoxin genes in Hiroshima prefecture. Jpn. J. Food Microbiol., 17,143-147 (2000). (in Japanese
12) Hyytiä, E., Björkroth, J., Hielm, S. and Korkeala , H.: Characterization of Clostridium botulinum groups I and II by randomly amplified polymorphic DNA analysis and repetitive element sequence-based PCR. Int. J. Food Microbiol., 48,179-189 (1999).
17) Midura, T. F. and Arnon, S. S.: Infant botulism: identification of Clostridium botulinum and its toxin in feces. Lancet, 2,934-936 (1976).
29) Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A. and Tingey, S. V.: DNA polymorphisms amplified by arbitrary primers are useful as genetic marker. Nucleic Acids Res., 18, 6531-6535 (1990).
6) Elmore, M. J., Hutson, R. A., Collins, M. D., Bodsworth , N. J., Whelan, S. M. and Minton, N. P.: Nucleotide sequence of the gene coding for proteolytic (group I) Clostridium botulinum type F neurotoxin: genealogi cal comparison with other clostridial neurotoxins. System. Appl. Microbiol., 18, 23-31 (1995).
24) Sakaguchi, G., Sakaguchi, S., Kamata, Y., Tabita, K., Asao, T. and Kozaki, S.: Distinct characters of Clostridium botulinum type A strains and their toxin associated with infant botulism in Japan. Int. J. Food Microbiol., 11,231-242 (1990).
18) Monma, C., Yanagawa, Y., Igarashi, H., Itoh, T., Kinpara, H., Aso, S. and Ohkawa, S.: Infant botulism case estimated by home-made vegetable soup in Tokyo. IASR, 17,241 (1996). (in Japanese
References_xml – reference: 19) Murmar, J.: A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol., 3,208-218 (1961).
– reference: 25) Shapiro, R. L., Hatheway, C. and Swerdlow, D. L.: Botulism in the United States: a clinical and epidemiological review. Ann. Intern. Med., 129,221-228 (1998).
– reference: 20) Nakano, H., Yoshikuni, Y., Hashimoto, H. and Sakaguchi, G.: Detection of Clostridium botulinum in natural sweetening. Int. J. Food Microbiol., 16,117-121 (1992).
– reference: 10) Henderson, I., Whelan, S. M., Davis, T. O. and Minton, N. P.: Genetic characterization of the botulinum toxin complex of Clostridium botulinum strain NCTC 2916. FEMS Microbiol. Lett., 140,151-158 (1996).
– reference: 28) Takeda, Y., Toukubo, Y., Inoue, K., Ogawa, H., Okada, S. and Sato, T.: Infant botulism by Clostridium botulinum possessing the type A and B botulism neurotoxin genes in Hiroshima prefecture. Jpn. J. Food Microbiol., 17,143-147 (2000). (in Japanese)
– reference: 2) Cordoba, J. J., Collins, M. D. and East, A. K.: Studies on the genes encoding botulinum neurotoxin type A of Clostridium botulinum from a variety of sources. System. Appl. Microbiol., 18, 13-22 (1995).
– reference: 11) Hutson, R. A., Collins, M. D., East, A. K. and Thompson, D. E.: Nucleotide sequence of the gene coding for non-proteolytic Clostridium botulinum type B neurotoxin: comparison with other clostridial neurotoxins. Curr. Microbiol., 28,101-110 (1994).
– reference: 16) Lund, B. M. and Peck, M. W.: Clostridium botulinum, the microbiological safety and quality of food. Lund, B. M., Baird-Parker, A. C., and Gould (eds.), p. 1057-1109, Aspen, Gaithersburg, Md. (2000).
– reference: 8) Franciosa, G., Floridi, F., Maugliani, A. and Aureli , P.: Differentiation of the gene clusters encoding botulinum neurotoxin type A complexes in Clostridium botulinum type A, Ab, and A (B) strains. Appl. Environ. Microbiol., 70, 7192-7199 (2004).
– reference: 9) Fujinaga, Y., Takeshi, K., Inoue , K., Fujita, R., Ohyama, T., Moriishi, K. and Oguma, K.: Type A and B neurotoxin genes in a Clostridium botulinum type AB strain. Biochem. Biophys. Res. Commun., 213,737-745 (1995).
– reference: 14) Kozaki, S., Nakaue, S. and Kamata, Y.: Immunological characterization of the neurotoxin produced by Clostridium botulinum type A associated with infant botulism in Japan. Microbiol. Immun., 39,767-774 (1995).
– reference: 22) Oguma, K., Inoue, K., Fujinaga, Y., Yokota, K. Watanabe, T., Ohyama, T., Takeshi, K. and Inoue, K.: Structure and function of Clostridium botulinum progenitor toxin. J. Toxicol.-Toxin Rev., 18, 17-34 (1999).
– reference: 3) East, A. K., Bhandari, M., Stacey, J. M., Campbell, K. D. and Collins, M. D.: Organization and phylogenetic interrelationships of genes encoding components of the botulinum toxin complex in proteolytic Clostridium botulinum types A, B, and F: evidence of chimeric sequences in the gene encoding the nontoxic nonhemagglutinin component. Int. J. Syst. Bacteriol., 46, 1105-1112 (1996).
– reference: 15) Kubota, T., Shirakawa, S., Kozaki, S., Isogai, E., Isogai, H., Kimura, K. and Fujii, N.: Mosaic type of the nontoxic- nonhemagglutinin component gene in Clostridium botulinum type A strain isolated from infant botulism in Japan. Biochem. Biophys. Res. Commun., 224,843-848 (1996).
– reference: 4) East, A. K. and Collins, M. D.: Conserved structure of genes encoding components of botulinum neurotoxin complex M and the sequence of the gene coding for the nontoxic component in nonproteolytic Clostridium botulinum type F. Curr. Microbiol., 29, 69-77 (1994).
– reference: 18) Monma, C., Yanagawa, Y., Igarashi, H., Itoh, T., Kinpara, H., Aso, S. and Ohkawa, S.: Infant botulism case estimated by home-made vegetable soup in Tokyo. IASR, 17,241 (1996). (in Japanese)
– reference: 26) Tabita, K., Sakaguchi, S., Kozaki, S. and Sakaguchi, G.: Comparative studies on Clostridium botulinum type A strains associated with infant botulism in Japan and in California, USA. Jpn. J. Med. Sci. Biol., 43,219-231 (1990).
– reference: 6) Elmore, M. J., Hutson, R. A., Collins, M. D., Bodsworth , N. J., Whelan, S. M. and Minton, N. P.: Nucleotide sequence of the gene coding for proteolytic (group I) Clostridium botulinum type F neurotoxin: genealogi cal comparison with other clostridial neurotoxins. System. Appl. Microbiol., 18, 23-31 (1995).
– reference: 13) Inoue, K., Fujinaga, Y., Watanabe, T., Ohyama , T., Takeshi, K., Moriishi, K., Nakajima, H., Inoue, K. and Oguma, K.: Molecular composition of Clostridium botulinum type A progenitor toxins. Infect . Immun., 64, 1589-1594 (1996).
– reference: 12) Hyytiä, E., Björkroth, J., Hielm, S. and Korkeala , H.: Characterization of Clostridium botulinum groups I and II by randomly amplified polymorphic DNA analysis and repetitive element sequence-based PCR. Int. J. Food Microbiol., 48,179-189 (1999).
– reference: 17) Midura, T. F. and Arnon, S. S.: Infant botulism: identification of Clostridium botulinum and its toxin in feces. Lancet, 2,934-936 (1976).
– reference: 29) Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A. and Tingey, S. V.: DNA polymorphisms amplified by arbitrary primers are useful as genetic marker. Nucleic Acids Res., 18, 6531-6535 (1990).
– reference: 23) Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Hoorn, G. T., Erlich, H. A. and Arnheim, N.: Enzymatic amplification of 13-globin genomic sequences and restriction site analysis for diagnosis of Siekle cell anemia. Science, 230, 1350-1354 (1985).
– reference: 27) Tabita, K., Sakaguchi, S., Kozaki, S. and Sakaguchi, G.: Distinction between Clostridium botulinum type A strains associated with food-borne botulism and those with infant botulism in Japan in intraintestinal toxin production in infant mice and some other properties. FEMS Microbiol. Lett., 79,251-256 (1991).
– reference: 7) Franciosa, G., Ferreira, J. L. and Hatheway , C. L.: Detection of type A, B, and E botulism neurotoxin genes in Clostridium botulinum and other Clostridium species by PCR: evidence of unexpressed type B toxin genes in type A toxigenic organisms. J. Clin. Microbiol., 32, 1911-1917 (1994).
– reference: 24) Sakaguchi, G., Sakaguchi, S., Kamata, Y., Tabita, K., Asao, T. and Kozaki, S.: Distinct characters of Clostridium botulinum type A strains and their toxin associated with infant botulism in Japan. Int. J. Food Microbiol., 11,231-242 (1990).
– reference: 5) East, A. K., Stacey, J. M. and Collins , M. D.: Cloning and sequencing of a hemagglutinin component of the botulinum neurotoxin complex encoded by Clostridium botulinum types A and B. System. Appl. Microbiol., 17,306-312 (1994).
– reference: 21) Oguma, K., Fujinaga, Y. and Inoue, K.: Structure and function of Clostridium botulinum toxins. Microbiol. Immunol., 39,161-168 (1995).
– reference: 1) Arnon, S. S.: Infant botulism. Ann. Rev. Med., 31,541-560 (1981).
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Snippet ボツリヌス菌の分子疫学解析手法として, RAPD法, 神経毒素複合体遺伝子を標的としたPCR法およびPCRRFLP法の有用性について検討した.試験菌株として, 食餌性ボツリヌス症由来株, 日本および米国の乳児ボツリヌス症由来株, 蜂蜜由来株など23株を用いた.RAPD法は,...
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Title RAPDおよびPCR-RFLP法によるA型毒素産生ボツリヌス菌の遺伝子型鑑
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ispartofPNX 日本食品微生物学会雑誌, 2005/12/30, Vol.22(4), pp.148-154
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