Loop-mediated isothermal amplification (LAMP) 法を用いた Vibrio vulnificus の迅速検出
Vibrio vulnificusは沿岸海水中に存在するグラム陰性桿菌で, 壊死性菌膜炎や敗血症の起炎菌である.感染者の多くは基礎疾患として肝機能障害や糖尿病等を有し, 本菌に汚染した生鮮魚介類の摂食, あるいは創傷部からの菌の侵入により感染する.病態は激烈で, 短期間で敗血症ショックを惹き起こし, 高い死亡率を呈することから, 早期診断・早期治療が必須であり本菌の迅速検出法の開発が求められている. Loop-mediated isothermal amplification (LAMP) 法は, 特定の遺伝子を迅速かつ高感度で検出する新しい遺伝子増幅法として注目されている.今回我々は, v....
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| Published in | 感染症学雑誌 Vol. 82; no. 5; pp. 407 - 413 |
|---|---|
| Main Authors | , , , , , |
| Format | Journal Article |
| Language | Japanese |
| Published |
一般社団法人 日本感染症学会
20.09.2008
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| Online Access | Get full text |
| ISSN | 0387-5911 1884-569X |
| DOI | 10.11150/kansenshogakuzasshi1970.82.407 |
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| Abstract | Vibrio vulnificusは沿岸海水中に存在するグラム陰性桿菌で, 壊死性菌膜炎や敗血症の起炎菌である.感染者の多くは基礎疾患として肝機能障害や糖尿病等を有し, 本菌に汚染した生鮮魚介類の摂食, あるいは創傷部からの菌の侵入により感染する.病態は激烈で, 短期間で敗血症ショックを惹き起こし, 高い死亡率を呈することから, 早期診断・早期治療が必須であり本菌の迅速検出法の開発が求められている. Loop-mediated isothermal amplification (LAMP) 法は, 特定の遺伝子を迅速かつ高感度で検出する新しい遺伝子増幅法として注目されている.今回我々は, v.vulnificusを検出するLAMP法を構築した.v.vulnificus28株及びv.vulnificus以外の24菌種52株を用いて特異性を確認したところ, 良好な判定結果を示した.また, 最小検出感度は, 反応液あたりlcfuであり, 極めて高感度での検出が可能であった. 更に臨床応用を想定し, 実験動物モデルの検体から鋳型DNAを調製する前処理法を検討した. 構築した簡便な前処理法とLAMP法を組み合わせることにより, 検体採取から判定まで1時間以内での検出が可能となった. |
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| AbstractList | Vibrio vulnificusは沿岸海水中に存在するグラム陰性桿菌で, 壊死性菌膜炎や敗血症の起炎菌である.感染者の多くは基礎疾患として肝機能障害や糖尿病等を有し, 本菌に汚染した生鮮魚介類の摂食, あるいは創傷部からの菌の侵入により感染する.病態は激烈で, 短期間で敗血症ショックを惹き起こし, 高い死亡率を呈することから, 早期診断・早期治療が必須であり本菌の迅速検出法の開発が求められている. Loop-mediated isothermal amplification (LAMP) 法は, 特定の遺伝子を迅速かつ高感度で検出する新しい遺伝子増幅法として注目されている.今回我々は, v.vulnificusを検出するLAMP法を構築した.v.vulnificus28株及びv.vulnificus以外の24菌種52株を用いて特異性を確認したところ, 良好な判定結果を示した.また, 最小検出感度は, 反応液あたりlcfuであり, 極めて高感度での検出が可能であった. 更に臨床応用を想定し, 実験動物モデルの検体から鋳型DNAを調製する前処理法を検討した. 構築した簡便な前処理法とLAMP法を組み合わせることにより, 検体採取から判定まで1時間以内での検出が可能となった. |
| Author | 中島, 幹夫 小島, 禎 永沢, 善三 根本, 二郎 草場, 耕二 大石, 浩隆 |
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| References | 14) Lee SE, Shin SH, Kim SY, Kim YR, Shin DH, Chung SS, et al.: vibrio vulnificus has the trans-membrane transcription activator ToxRS stimulating the expression of the hemolysin gene vvhA. J Bacteriol 2000; 182: 3405-15. 5) Mori Y, Nagamine K, Tomita N, Notomi T: Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Biophys. Res. Commun 2001; 289: 150-4. 7) Hong TC, Mai QL, Cuong DV, Parida M, Minekawa H, Notomi T, et al.: Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acutere spiratory syndrome coronavirus. J Clin Microbio1 2004; 42: 1956-61. 3) Oishi H, Ura Y, Mitsumizo S, Nakashima M: A collective review of Vibrio vulnificus infection in Japan. Kansenshogaku Zasshi 2006; 80: 680-9. 15) Peeler JT, Houghtby GA, Rainosek AP: The most probable number technique in Compen-dium of methods for the microbiological examination of food. American Public Health Associa-tion, Washington, D. C., 1992; p.105-20. 20) 井上浩章, 野田晃子, 縣邦雄安中敏光, 小島禎, 渡一, 他: LAMP法, PCR法を用いた浴槽水レジオネラ属菌の迅速検査に関する調査研究. 防菌防徽 2004; 32: 481-7. 16) Hill WE, Keasler SP, Trucksess MW, Feng P, Kaysner CA, Lampel KA: Polymerase chain re-action identification of vibrio vulnificus in artifi-cially contaminated oysters. Appl Environ Mi-crobiol 1991; 57: 707-11. 17) Hara-Kudo Y, Miwa N, Yamasaki S, Yatsuyan-agi J, Iwade Y, Takahash H, et al.: Quantitative detection of Vibrio vulnificus in seafood. Kansen-shogaku Zasshi 2005; 79: 931-6. 19) Takahashi H, Hara-Kudo Y, Miyasaka J, Kumagai S, Konuma H: Development of a quantitative real-time polymerase chain reaction targeted to the tox R for detection of vibrio vulnificus. J Microbiol Methods 2005; 61: 77-85. 4) Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, eta1.: Loop-mediated isothermal amplification of DNA. Nucleic Acid Res 2000; 28: e63. 11) Finegold SM, Martin WJ: Microorganisms encountered in the blood. In: Diagnostic microbiology 6th edition. The C. V. Mosby Company, St. LOUIS, 1982; p.40-64. 13) 岩倉信弘, 小塩精一, 中山照子, 山本達男: vibrio vulnificus の病原性の解析 (2): 感染動物モデル footpad 法の開発とカスパーゼ活性化. 感染症誌 2004; 78 (臨時増刊号): 146. 1) 古城八寿子, 城野昌義中川敬一, 小野友道: Vibrio vulnificus 感染症チャートの試み. 日皮会誌 1999; 109: 875-84. 18) Panicker G, Myers ML, Bej AK: Rapid detection of Vibrio vulnificus in shellfish and Gulf of Mexico water by real-time PCR. Appl Environ Microbiol 2004; 70: 498-507. 9) Hara-Kudo Y, Ohtsuka K, Segawa Y, Takatori K, Kojima T, Ikedo M, et al.: Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification. J Med Microbiol 2007; 56: 398-406. 12) Finegold SM, Martin WJ: Microorganisms encountered in wounds, abscesses, and bite infections; anaerobic procedures. In: Diagnostic microbiology 6th edition. The C. V. Mosby Company, St. LOUIS, 1982; p.128-42. 10) Nagamine K, Hase T, Notomi T: Accelerated reaction by loop-mediated isothermal amplifica-tion using loop primers. Mol Cell Probes 2002; 16: 223-9. 2) Fujisawa N, Yamada H, Kohda H, Tadano J, Hayashi S: Necrotizing fasciitis caused by Vibrio vulnificus differs from that caused by strep-tococcal infection. J Infect 1998; 36: 313-6. 6) Kojima T, Shibata N, Ikedo M, Arakawa Y: Development and evaluation of novel loop-mediated isothermal amplification for rapid detection of bla (IMP-1) and bla (VIM-2) genes. Kansenshogaku Zasshi 2006; 80: 405-12. 8) Parida M, Posadas G, Inoue S, Hasebe F, Morita K: Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus. J Clin Microbiol 2004; 42: 257-63. |
| References_xml | – reference: 4) Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, eta1.: Loop-mediated isothermal amplification of DNA. Nucleic Acid Res 2000; 28: e63. – reference: 12) Finegold SM, Martin WJ: Microorganisms encountered in wounds, abscesses, and bite infections; anaerobic procedures. In: Diagnostic microbiology 6th edition. The C. V. Mosby Company, St. LOUIS, 1982; p.128-42. – reference: 2) Fujisawa N, Yamada H, Kohda H, Tadano J, Hayashi S: Necrotizing fasciitis caused by Vibrio vulnificus differs from that caused by strep-tococcal infection. J Infect 1998; 36: 313-6. – reference: 3) Oishi H, Ura Y, Mitsumizo S, Nakashima M: A collective review of Vibrio vulnificus infection in Japan. Kansenshogaku Zasshi 2006; 80: 680-9. – reference: 8) Parida M, Posadas G, Inoue S, Hasebe F, Morita K: Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus. J Clin Microbiol 2004; 42: 257-63. – reference: 6) Kojima T, Shibata N, Ikedo M, Arakawa Y: Development and evaluation of novel loop-mediated isothermal amplification for rapid detection of bla (IMP-1) and bla (VIM-2) genes. Kansenshogaku Zasshi 2006; 80: 405-12. – reference: 19) Takahashi H, Hara-Kudo Y, Miyasaka J, Kumagai S, Konuma H: Development of a quantitative real-time polymerase chain reaction targeted to the tox R for detection of vibrio vulnificus. J Microbiol Methods 2005; 61: 77-85. – reference: 13) 岩倉信弘, 小塩精一, 中山照子, 山本達男: vibrio vulnificus の病原性の解析 (2): 感染動物モデル footpad 法の開発とカスパーゼ活性化. 感染症誌 2004; 78 (臨時増刊号): 146. – reference: 17) Hara-Kudo Y, Miwa N, Yamasaki S, Yatsuyan-agi J, Iwade Y, Takahash H, et al.: Quantitative detection of Vibrio vulnificus in seafood. Kansen-shogaku Zasshi 2005; 79: 931-6. – reference: 14) Lee SE, Shin SH, Kim SY, Kim YR, Shin DH, Chung SS, et al.: vibrio vulnificus has the trans-membrane transcription activator ToxRS stimulating the expression of the hemolysin gene vvhA. J Bacteriol 2000; 182: 3405-15. – reference: 7) Hong TC, Mai QL, Cuong DV, Parida M, Minekawa H, Notomi T, et al.: Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acutere spiratory syndrome coronavirus. J Clin Microbio1 2004; 42: 1956-61. – reference: 1) 古城八寿子, 城野昌義中川敬一, 小野友道: Vibrio vulnificus 感染症チャートの試み. 日皮会誌 1999; 109: 875-84. – reference: 20) 井上浩章, 野田晃子, 縣邦雄安中敏光, 小島禎, 渡一, 他: LAMP法, PCR法を用いた浴槽水レジオネラ属菌の迅速検査に関する調査研究. 防菌防徽 2004; 32: 481-7. – reference: 11) Finegold SM, Martin WJ: Microorganisms encountered in the blood. In: Diagnostic microbiology 6th edition. The C. V. Mosby Company, St. LOUIS, 1982; p.40-64. – reference: 15) Peeler JT, Houghtby GA, Rainosek AP: The most probable number technique in Compen-dium of methods for the microbiological examination of food. American Public Health Associa-tion, Washington, D. C., 1992; p.105-20. – reference: 18) Panicker G, Myers ML, Bej AK: Rapid detection of Vibrio vulnificus in shellfish and Gulf of Mexico water by real-time PCR. Appl Environ Microbiol 2004; 70: 498-507. – reference: 10) Nagamine K, Hase T, Notomi T: Accelerated reaction by loop-mediated isothermal amplifica-tion using loop primers. Mol Cell Probes 2002; 16: 223-9. – reference: 16) Hill WE, Keasler SP, Trucksess MW, Feng P, Kaysner CA, Lampel KA: Polymerase chain re-action identification of vibrio vulnificus in artifi-cially contaminated oysters. Appl Environ Mi-crobiol 1991; 57: 707-11. – reference: 9) Hara-Kudo Y, Ohtsuka K, Segawa Y, Takatori K, Kojima T, Ikedo M, et al.: Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification. J Med Microbiol 2007; 56: 398-406. – reference: 5) Mori Y, Nagamine K, Tomita N, Notomi T: Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Biophys. Res. Commun 2001; 289: 150-4. |
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| Snippet | Vibrio vulnificusは沿岸海水中に存在するグラム陰性桿菌で, 壊死性菌膜炎や敗血症の起炎菌である.感染者の多くは基礎疾患として肝機能障害や糖尿病等を有し, 本菌に汚... |
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| Title | Loop-mediated isothermal amplification (LAMP) 法を用いた Vibrio vulnificus の迅速検出 |
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