JP2013524814A - 試料からの低濃度の細菌の抽出 - Google Patents
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Abstract
Description
本願は、2010年4月21日に出願された米国仮特許出願第61/326,588号の利益および優先権を主張し、この米国仮特許出願の全体の内容は、本明細書中に参考として援用される。
本発明は、試料について、試料から細菌を単離するアッセイを実行することに一般に関し、そこで、アッセイは試料中の約1CFU/mlという低い濃度の細菌を単離する。
血液由来病原体は、健康管理上重要な問題である。細菌感染症の診断の遅れまたは不適切な診断は、感染への重大でしばしば致命的な炎症性応答である敗血症をもたらすことがある。敗血症は、米国で10番目の主要な死因である。血液中の細菌感染症の早期発見は、敗血症の開始の阻止での鍵である。血液由来感染症の伝統的な検出および同定の方法には、血液培養および抗生物質感受性アッセイが含まれる。それらの方法は細胞を培養することを一般に必要とし、それは費用がかかり、72時間という長い時間を要することがある。細胞培養結果が得られる前に敗血症性ショックが起こることがしばしばある。
本発明は、生物試料中の病原体を単離するための方法およびデバイスを提供する。本発明は、試料中の非常に低いレベルでの病原体の急速な検出を可能にし、したがって、病原体の早期発見および正確な検出および同定を可能にする。特定の態様では、本発明は、標的特異的結合部分を有する磁気粒子を用いて実施される。本発明の方法は、標的特異的結合部分を含む磁気粒子を体液試料に導入して混合物を作製すること、この混合物をインキュベートして上記粒子を標的に結合させること、磁場を加えて表面上に標的/磁気粒子複合体を捕捉すること、および粒子凝集を低減する洗浄溶液で洗浄し、それによって標的/磁気粒子複合体を単離することを含むことができる。本発明の方法の特別な利点は、多くの臨床試料に存在する低い濃度(血液試料に1CFU/mlという低い濃度の細菌)での、血液試料からの細菌および真菌の直接的な捕捉および単離についてのものである。
本発明は、試料から細菌を単離するアッセイを試料で実行することに一般に関し、そこで、アッセイは試料中の1CFU/mlという低い濃度の細菌を単離する。試料中の1CFU/mlという低い濃度の細菌を単離することができる任意のアッセイを用いることができる。ある態様では、本発明の方法は、体液試料中の標的病原体を捕捉する特定の磁気モーメントを有する磁気粒子(粒径および磁性材料の重量%で決定される)、および標的を単離するための磁石を用いて、試料中の1CFU/mlという低い濃度の細菌を単離する。
この開示全体で、特許、特許出願、特許公報、雑誌、本、論文、ウェブコンテンツなどの他の文書が参照および引用されている。全てのそのような文書は、全ての目的のためにここに参照により完全に本明細書に組み込まれる。
本明細書で示され、記載されるものに加えて、本発明およびその多くのさらなる実施形態の様々な変更は、本明細書で引用される科学文献および特許文献への参照を含むこの文書の全内容から、当業者に明らかになる。本明細書の対象物は、その様々な実施形態および同等物での本発明の実施へ適合させることができる、重要な情報、例証および指針を含む。
健康なボランティアからの血液試料に、血流感染症で最も頻繁に見出される細菌種の研究所株および臨床分離物の両方を含む細菌の臨床上の適切な濃度(1〜10CFU/mL)を混入させた。
ポリクローナルの汎グラム陽性細菌特異的IgGを生み出すために、最初に完全フロイントアジュバントに懸濁させた細菌抗原をリンパ節内に投与し、続いて不完全フロイントアジュバント中の細菌抗原の皮下注射を2週間隔で行うことによってヤギを免疫化した。細菌を指数増殖期(OD600=0.4〜0.8)まで増殖させることによって、抗原を抗体産生のために調製した。遠心による細菌の集菌の後、37℃で4時間の4%ホルムアルデヒドでのホルマリン固定を用いて、細菌を不活性化した。PBSで細菌を3回洗浄した後(15分間洗浄、4000rpmで20分間の遠心)、BCAアッセイを用いて抗原濃度を測定し、免疫化のために抗原を1mg/mLで用いた。グラム陽性細菌特異的IgGを生み出すために、接種のためにいくつかの細菌種を用いた:Staphylococcus aureus、Staphylococcus epidermidis、Enterococcus faeciumおよびEnterococcus fecalis。
超常磁性ビーズは、酸化鉄ナノ粒子(直径5〜15nm)をラテックスコアに封入し、ヤギIgGで標識することによって合成された。有機溶媒にナノ粒子を含む強磁性流体をエタノールで沈殿させ、スチレンおよび界面活性剤Hitenol BC−10の水溶液にナノ粒子を再懸濁させ、超音波処理を用いて乳化した。混合液を撹拌させながら一晩平衡させ、1.2および0.45μmフィルターによって濾過して、均一なミセルサイズを達成した。スチレン、アクリル酸およびジビニルベンゼンを、pH9.6の炭酸バッファーに加えた。K2S2O8の添加により70℃の混合液で重合を開始させ、反応は一晩完了させた。合成された粒子は、磁気捕捉を用いて0.1%SDSで3回洗浄し、1.2、0.8および0.45μmフィルターによって濾過し、抗体コンジュゲーションのために用いた。
血流感染の間に血液に存在する細菌を、上の実施例3で調製される超常磁性ビーズを用いて磁気標識した。実施例1に記載の混入された試料をトリスベースの結合バッファーおよび標的特異的ビーズで3倍に希釈し、その後37℃の振盪プラットホーム上で最大2時間インキュベートした。インキュベーションの後、標識標的を磁気によって分離し、その後血液生成物を除去するように設計された洗浄工程を行った。下の実施例5を参照されたい。
磁気標識標的細菌および過量の遊離ビーズを含む血液を、試料の流れに直角に置かれたいくつかの強力な希土類棒磁石を有するフロースルー捕捉セルに注入した。流路断面0.5mm×20mm(h×w)および7個のNdFeB棒磁石を有するフローチャンバーを用いて、5mL/分という速さの流速が達成された。磁石の存在下で混合液をチャンネル内に流した後に、ヘパリンを含む洗浄溶液をチャンネル内に流した。血液成分を除去して磁気粒子の凝集体の形成を低減するために、結合した標的をヘパリン含有バッファーで1回洗浄した。結合した標的を効果的に洗浄するために、磁石を取り出し、捕捉された磁性材料を洗浄バッファーに再懸濁させ、その後磁場を再び加え、同じフロースルー捕捉セルで磁性材料を捕捉した。
Claims (25)
- 試料から細菌を単離するための方法であって、
細菌を含む試料を得る工程と;
前記試料について、前記試料から前記細菌を単離するアッセイを実行する工程であって、前記アッセイは、前記試料中の約1CFU/mlという低い濃度の細菌を単離する工程と
を含む、方法。 - 前記試料がヒトの組織または体液である、請求項1に記載の方法。
- 前記体液が血液である、請求項2に記載の方法。
- 前記細菌が血液由来細菌である、請求項3に記載の方法。
- 前記試料が食物試料である、請求項1に記載の方法。
- 前記細菌が食物由来細菌である、請求項5に記載の方法。
- 前記細菌を特徴づける工程をさらに含む、請求項1に記載の方法。
- 特徴づける工程が前記細菌を同定する工程を含む、請求項7に記載の方法。
- 同定する工程が前記細菌に由来する核酸を配列決定する工程、または前記細菌に由来する核酸を増幅する工程から選択される、請求項8に記載の方法。
- 前記細菌がグラム陽性細菌である、請求項1に記載の方法。
- 前記細菌がグラム細菌である、請求項1に記載の方法。
- 試料から細菌を単離するための方法であって、
細菌を含む試料を得る工程と;
前記試料から前記細菌を単離するために磁気粒子を用いてアッセイを行う工程であって、前記磁気粒子は、前記アッセイが前記試料中の約1CFU/mlという低い濃度の細菌を単離するようなサイズであり、かつそのような量の磁性材料を含む工程と
を含む、方法。 - 前記磁気粒子が細菌特異的結合部分を含む、請求項12に記載の方法。
- 前記細菌特異的結合部分が抗体である、請求項13に記載の方法。
- 前記試料がヒトの組織または体液である、請求項12に記載の方法。
- 前記体液が血液である、請求項15に記載の方法。
- 前記細菌が血液由来細菌である、請求項16に記載の方法。
- 前記試料が食物試料である、請求項12に記載の方法。
- 前記細菌が食物由来細菌である、請求項18に記載の方法。
- 前記細菌を特徴づける工程をさらに含む、請求項12に記載の方法。
- 特徴づける工程が、前記細菌を同定する工程を含む、請求項20に記載の方法。
- 同定する工程が、前記細菌に由来する核酸を配列決定する工程、または前記細菌に由来する核酸を増幅する工程から選択される、請求項21に記載の方法。
- 前記細菌がグラム陽性細菌である、請求項12に記載の方法。
- 前記細菌がグラム細菌である、請求項12に記載の方法。
- 試料から細菌を単離するための方法であって、
細菌を含む試料を得る工程と;
前記試料から前記細菌を単離するために磁気粒子を用いてアッセイを行う工程であって、前記磁気粒子は、前記試料中の約1CFU/mlという低い濃度の細菌の単離を可能にする磁気モーメントを有する工程と
を含む、方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US32658810P | 2010-04-21 | 2010-04-21 | |
US61/326,588 | 2010-04-21 | ||
PCT/US2011/033410 WO2011133759A1 (en) | 2010-04-21 | 2011-04-21 | Extractin low concentrations of bacteria from a sample |
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Application Number | Title | Priority Date | Filing Date |
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JP2016198681A Division JP2017012198A (ja) | 2010-04-21 | 2016-10-07 | 試料からの低濃度の細菌の抽出 |
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JP2013524814A true JP2013524814A (ja) | 2013-06-20 |
JP2013524814A5 JP2013524814A5 (ja) | 2014-06-19 |
JP6023698B2 JP6023698B2 (ja) | 2016-11-09 |
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