JP6563654B2 - 光経路により生体試料中の微生物を検出し直接同定する方法 - Google Patents
光経路により生体試料中の微生物を検出し直接同定する方法 Download PDFInfo
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Description
・ 調査が原材料、中間体、及び市販最終製品に適用される、病原性微生物とその毒素
・ 原材料から最終製品まで、チェーンを通じて生産工程の品質の指標として使用される非病原性微生物、及び
・ 酵素などの技術的対象となる細菌
の間で区別がなされる。
a)第1の容器において、前記試料を少なくとも1つの培地と接触させる工程と、
b)前記第1の容器を適切な条件に置いて微生物の増殖を可能にする工程と、
c)前記第1の容器又は第2の容器中の前記微生物を捕捉するための反応混合物及び基質であって、前記微生物を検出する手段を含む前記反応混合物と、試料及び培地からなる混合物の一部又は全てを接触させる工程と、
d)前記第1又は第2の容器内で、検出手段により検出され捕捉基質に固定された微生物の存在を検出する工程と
を本質的に含む方法に関する。
この実験の目的は、Nunc/Thermo Scientific社(カタログ番号472230)により市販され、図2及び3に示されている照射ポリスチレン製感作基質を用いた、反応混合物での継代培養における食品試料中の標的細菌サルモネラ・ナポリの存在の直接検出である。
工程1:一次増菌培地への試料の再懸濁
2つの試料は次のように調製する:
試料A:ホモジナイズバッグにおいて、5コロニー形成単位(CFU)のサルモネラ・ナポリに汚染した25gの非低温殺菌牛乳チーズを、1mlのSupplement SPT(ビオメリュー、照会番号42650)を補充した225mLの緩衝ペプトン水(BPW)(ビオメリュー、照会番号42043)に再懸濁する;
試料Aホモジナイズバッグからの0.1mLを、10mLのSX2(ビオメリュー、照会番号42121)及び1.6g/LのTTC(ビオメリュー、カタログ番号04568088)を含有する反応チューブに移す。これを試料A’とする。
感作捕捉基質を各チューブに入れる(試料A’及びB’)。次いでチューブを再び密閉し、37℃で6時間、ストーブ中でインキュベートする。
インキュベーション(37℃で6時間)の終わり、及び試料中に存在する細菌の全て(すなわち、追加の細菌叢及び標的細菌叢に属する)によるTTCの非特異的還元後、反応混合物は赤くなった。故に、捕捉基質を観察し、分析試料が陽性又は陰性かどうかを明らかにするため、反応混合物から前記捕捉基質を単離するようにチューブを傾ける。
この実験の目的は、Nunc/Thermo Scientific社(カタログ番号472230)により市販され、図2及び3に示されている照射ポリスチレン製感作基質を用いた、増菌食品試料中の標的細菌サルモネラ・ナポリの存在の直接検出である。
工程1:一次増菌培地への試料の再懸濁
2つの試料は次のように調製する:
試料A:ホモジナイズバッグにおいて、サルモネラ・ナポリに汚染した25gのミンチステーキを、1mLのSupplement SPT(ビオメリュー、照会番号42650)を補充した225mLのBPW(ビオメリュー、照会番号42043)に再懸濁する。
試料Aホモジナイズバッグからの1mLを、10μLのゲンチアナバイオレット(ビオメリュー、照会番号55545)を補充した1mLのトリプトン塩(ビオメリュー、照会番号42076)を含有する反応チューブに移す。これを試料A’とする。
以下に記載の通り、感作捕捉基質を各チューブに入れる(試料A’及びB’)。次いで反応期間中、チューブを再び密閉する。
反応(室温で40分)の最後に、試料中に存在する細菌の全て(すなわち、追加の細菌叢及び標的細菌叢に属する)が紫に染色される。故に、捕捉基質を観察できるようにし、分析試料が陽性又は陰性かどうかを明らかにするため、反応混合物から前記捕捉基質を単離するようにチューブを傾ける。
Claims (11)
- 試料中に存在する少なくとも1つの微生物を検出する方法において、
a)第1の容器において、前記試料を少なくとも1つの培地と接触させる工程と、
b)前記第1の容器を適切な条件で6−48時間培養し、前記微生物の増殖を可能にする工程と、
c)工程b)の後に、前記第1の容器又は第2の容器中で前記微生物を捕捉するための単一片基質及び反応混合物であって、前記微生物を検出する手段を含む前記反応混合物と、前記試料及び前記培地からなる混合物の一部又は全てを接触させる工程と、
d)混合物を含む前記第1又は第2の容器内で、前記検出する手段により検出され前記捕捉するための単一片基質に固定された前記微生物の存在を検出する工程と
を本質的に含む方法。 - 前記第1又は第2の容器を適切な条件に置いて前記微生物の増殖を可能にすることからなる中間工程c’)を含む、請求項1に記載の方法。
- 検出された前記微生物の検出を確証することからなる追加の工程e)を含む、請求項1又は2に記載の方法。
- 前記追加の工程e)が、検出に使用された前記検出する手段と同一の又は異なる検出する手段を用いて実施される、請求項3に記載の方法。
- 前記微生物の少なくとも1つの特異的又は非特異的結合パートナーが前記捕捉するための単一片基質に固定される、請求項1から4のいずれか一項に記載の検出の方法。
- 前記特異的結合パートナーが、抗体、Fab断片、Fab’断片、アプタマー、組換え又は非組換えファージタンパク質、ファージを含む群から採られる、請求項5に記載の検出の方法。
- 前記微生物の検出がリアルタイムで実施される、請求項1から6のいずれか一項に記載の方法。
- 前記微生物の検出が、前記微生物の前記増殖を可能にする工程の最後に実施される、請求項1から7のいずれか一項に記載の検出の方法。
- 前記第1及び/又は第2の容器が、ホモジナイズバッグ、フラスコ、ボトル又はタブレット容器である、請求項1から8のいずれか一項に記載の検出の方法。
- 前記捕捉するための単一片基質が、多孔質の単一片基質である、請求項1から9のいずれか一項に記載の方法。
- 前記捕捉するための単一片基質が保護膜により保護される、請求項10に記載の方法。
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FR1250249 | 2012-01-10 | ||
FR1250249A FR2985519B1 (fr) | 2012-01-10 | 2012-01-10 | Procede de detection et d'identification directe par voie optique d'un microorganisme dans un echantillon biologique |
PCT/FR2013/050051 WO2013104864A1 (fr) | 2012-01-10 | 2013-01-09 | Procede de detection et d'identification directe par voie optique d'un microorganisme dans un echantillon biologique |
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JP2015504163A JP2015504163A (ja) | 2015-02-05 |
JP6563654B2 true JP6563654B2 (ja) | 2019-08-21 |
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US (1) | US9902987B2 (ja) |
EP (1) | EP2802875B1 (ja) |
JP (1) | JP6563654B2 (ja) |
CN (1) | CN104115010A (ja) |
ES (1) | ES2875894T3 (ja) |
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FR2962445B1 (fr) * | 2010-07-08 | 2013-06-28 | Biomerieux Sa | Procede de detection et d'identification directe d'un microorganisme dans un echantillon biologique dilue dans un bouillon d'enrichissement |
FR2997703B1 (fr) * | 2012-11-07 | 2016-12-30 | Biomerieux Sa | Procede de traitement d'au moins un echantillon biologique |
EP3280792A1 (en) * | 2015-04-08 | 2018-02-14 | 3M Innovative Properties Company | Stand-up gusseted filter bag |
KR102050166B1 (ko) * | 2018-08-10 | 2019-11-28 | 연세대학교 산학협력단 | 부유 미생물의 실시간 연속 측정장치 |
FR3086952A1 (fr) * | 2018-10-08 | 2020-04-10 | Biomerieux | Procede de prelevement de microorganismes a partir d'un echantillon biologique liquide ou visqueux ou d'une suspension de microorganismes fortement contamines |
JP7344540B2 (ja) * | 2019-06-19 | 2023-09-14 | 株式会社Icst | 試験器具および試験方法 |
IT202100003080A1 (it) * | 2021-02-11 | 2022-08-11 | Mectron S P A | Un metodo per la cattura di batteri da sospensioni e relativo dispositivo |
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US5094955A (en) * | 1988-03-15 | 1992-03-10 | Akzo N.V. | Device and method for detecting microorganisms |
AU705576B2 (en) * | 1995-06-05 | 1999-05-27 | Biomerieux, Inc. | Device and method for detecting microorganisms |
US5843699A (en) * | 1997-04-08 | 1998-12-01 | Difco Laboratories, Inc. | Rapid microorganism detection method |
JP3773633B2 (ja) * | 1997-10-31 | 2006-05-10 | 株式会社三菱化学ヤトロン | 大腸菌o157の分析方法及び分析用試薬 |
ATE449339T1 (de) * | 2000-11-30 | 2009-12-15 | Boston Probes Inc | Verfahren und zusammensetzungen zum aussortieren und/oder nachweis von mikroorganismen |
FR2819266B1 (fr) * | 2001-01-05 | 2004-01-16 | Alain Rambach | Milieu de culture pour la detection et/ou la discrimination des bacteries du genre listeria et procede de mise en oeuvre |
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CA2462855C (en) | 2001-10-10 | 2011-09-13 | Tecra International Pty Ltd | System and apparatus for use in detecting microorganisms |
AU2007229320B2 (en) * | 2006-03-17 | 2013-01-10 | Newsouth Innovations Pty Limited | Electrochemical sensor |
JP4339375B2 (ja) * | 2007-05-21 | 2009-10-07 | 白鶴酒造株式会社 | 微生物センサーおよびその製造方法 |
FR2928656B1 (fr) * | 2008-03-14 | 2011-08-26 | Biomerieux Sa | Procede de detection en temps reel de microorganismes dans un milieu de culture liquide par agglutination. |
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FR2962445B1 (fr) * | 2010-07-08 | 2013-06-28 | Biomerieux Sa | Procede de detection et d'identification directe d'un microorganisme dans un echantillon biologique dilue dans un bouillon d'enrichissement |
CN203007262U (zh) * | 2013-01-15 | 2013-06-19 | 温州医学院 | 微阵列细菌识别和筛分装置 |
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FR2985519A1 (fr) | 2013-07-12 |
EP2802875B1 (fr) | 2021-03-31 |
FR2985519B1 (fr) | 2016-02-19 |
US9902987B2 (en) | 2018-02-27 |
WO2013104864A1 (fr) | 2013-07-18 |
CN104115010A (zh) | 2014-10-22 |
US20140349280A1 (en) | 2014-11-27 |
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