JP5460866B2 - 敗血症の質量分析法診断 - Google Patents
敗血症の質量分析法診断 Download PDFInfo
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Description
最も単純な方法においては、選択したコロニーから微生物の一部を、小さなスワブを用いて質量分析試料支持体に移し、マトリックス支援レーザー脱離イオン化法(MALDI)のために、従来のマトリックス物質を強酸性にした溶液(通常はα-シアノ-4-ヒドロキシ桂皮酸(HCCA)または2,5-ジ-ヒドロキシ-安息香酸(DHB))を撒く。この酸(通常は蟻酸またはトリフルオロエタン酸)が細胞壁を攻撃し、マトリックス溶液の有機溶媒(通常はアセトニトリル)が微生物細胞内に侵入して、細胞壁を弱らせ、破裂させる。試料は次に、溶媒を蒸発させることにより乾かし、これにより、溶解していたマトリックス物質が結晶化する。可溶性タンパク質と、それより少量ながら細胞内の他の物質が、マトリックス結晶内に埋め込まれる。
この目的には通常、特殊なMALDI飛行時間質量分析計(MALDI-TOF-MS)が使用される。マススペクトルは、これら分析対象イオンの質量値のプロフィールである。これらは主にタンパク質イオンであり、およそ3,000ダルトン〜15,000ダルトンの質量を有する最も有用な情報を備えたイオンである。この方法のタンパク質イオンは主に1価のみ(電荷数z=1)であり、これにより、常に用語「質量電荷比」m/z(これは他のタイプの質量分析では実際に必要であり慣習的である)を使用する代わりに、イオンの質量mを単純に参照することができる。
48個、96個、または384個の試料スポットをそれぞれ有する質量分析試料支持体は市販されている。これら数多くの試料からマススペクトルを取得するには30分〜2時間かかる。緊急に同定が必要な場合は、個々の微生物試料が培養後数分で同定できる(培養後であっても、これは常に時間のかかる作業である)。
これらはすべて、衝撃により二次電子を生成するからである。ある種の親イオンからのイオン源が加速した後に生成されるフラグメントイオンおよび中性粒子はすべて、親イオンと同じ速度を有するため、同時にイオン検出器に到達する。この到達時間が、分割されていない元のイオンの質量の測定値である。
個別スペクトルそれぞれのイオンは、各スペクトルの紫外線パルスレーザーのレーザーフラッシュ1回によって生成される。個別スペクトルの測定ダイナミックレンジが低く、信号のノイズが高いため、総和スペクトルはこの方法で生成されなければならない。少なくとも約50、場合によっては1000を超える個別スペクトルがここで取得される。総和スペクトルは一般に、最新の質量分析計によって取得され数秒以内に集積された数百の個別スペクトルから成っている。
最良の同定結果は、3,000〜15,000ダルトンの質量範囲における質量信号のみが評価される場合に得られる。所定の条件によって起こり得るように、質量分解能が低いということは、1ダルトンずつ異なる質量信号を有する同位体群は、この質量範囲では解決されないことがあり得る。質量信号はよって、同位体群の範囲の形状を反映したものになる。
前記文書に記述されているように、この微生物を遠心分離またはマイクロフィルターで沈殿させる方法は、直接適用することができ、リンパ液、滑液、脳脊髄液、更には尿や涙液などの分泌体液にも高い成功率を収めている。例えば全血のように、内因性血球を含んでいる体液については、最初に血液を培養することによって微生物を成長させる中間ステップを導入する必要がある。これは、微生物の濃度が通常、1ミリリットル当たり0.5〜10個の範囲でしかなく、赤血球や白血球などの血球を完全かつ徹底的に破壊してヒトのタンパク質をすべて完全に排除してからとなる。上述の文書において、この破壊は、具体的に蒸留水の添加により実施されている。ヒトの血球の細胞膜は非常にデリケートで、水侵入の浸透圧により容易に破壊される。これは細胞の細胞壁がはるかに強固であるのに比べ対照的である。蒸留水を加えて遠心分離することを繰り返すことにより、十分にクリーンなペレットが得られ、これにはヒト血球の残留物はもはや含まれない。
稀少かつ難しい疾患を有する患者を擁するそのような種類の病院では、最高40パーセントの血液試料が、SDS溶液で処理した後、深赤色液中に隠れて浮遊する大きな粘液塊を形成する。これらの粘液プラグは、未知の割合の微生物を抱き込んでいる。熟練した当業者は、更なる治療のために、プラグ周囲から液の一部をピペットで採取することができるが、この方法は難しく、未知の要素により検出限度が低下する。ある研究では、これらの粘液塊は主に過剰に増加した白血球のDNAを含んでおり、おそらくは血液の凝固したタンパク質と混合していることが示唆されている。患者は通常、血中の白血球数の非常な増加を示している。
この深赤色の沈殿は、微生物だけでなく、1ミリリットルの血液に由来する500万個の赤血球、7000個の白血球、20万個の血小板を通常含んでおり、振盪器内で追加された界面活性剤と混合されることにより、血球の細胞膜が破壊され、可溶性タンパク質が放出される。この深赤色溶液を再び遠心分離にかけ、今度は上澄みが深赤色のままとなり、沈殿はもし見える場合は純白となる。この上澄みを除去し、界面活性剤溶液を充填し、遠心分離にかけるプロセスを、ここで繰り返して行い、血球の最後の残留物すらも除去することができる。このプロセスを純蒸留水を用いて繰り返すことにより、界面活性剤を除去することができる。この界面活性剤は、マトリックス支援レーザー脱離イオン化法(MALDI)を阻害する可能性があるためである。上澄みを最後に除去した後、その沈殿物は、目に見えるものであるか否かを問わず、上述の方法で分解され、可溶性タンパク質は試料支持プレートに移される。
Claims (17)
- 血液中にある、細菌または酵母を含む微生物を、遠心分離または濾過によって沈殿させ、該微生物のマススペクトルを取得し、該マススペクトルによって該微生物を同定する質量分析同定の方法であって、
該微生物の沈殿の前に、界面活性剤を添加することによって、血液培養からの血液試料の血球を溶解させることを特徴とする、方法。 - 前記血液中の十分な数の微生物が、培養によって産生される、請求項1に記載の方法。
- 遠心分離によって沈殿した前記微生物を洗浄し、再び遠心分離にかける、請求項1または請求項2に記載の方法。
- 前記界面活性剤としてドデシル硫酸ナトリウム(SDS)が使用される、請求項1〜請求項3のいずれか1項に記載の方法。
- 前記界面活性剤が溶液として添加される、請求項1〜請求項4のいずれか1項に記載の方法。
- 前記界面活性剤溶液が消泡剤を含む、請求項5に記載の方法。
- 前記血液試料に抗凝固剤が添加される、請求項1〜請求項6のいずれか1項に記載の方法。
- 前記血液試料に1つ以上のヌクレアーゼが添加される、請求項1〜請求項7のいずれか1項に記載の方法。
- 前記血液試料のための前記血液が、蒸留水で1:2〜1:10の希釈率で希釈される、請求項1〜請求項8のいずれか1項に記載の方法。
- 前記血液試料のための前記血液が、蒸留水で約1:5の希釈率で希釈される、請求項9に記載の方法。
- 前記血液試料が遠心分離され、その上澄みを除去してから、前記界面活性剤溶液が添加される、請求項5または請求項6に記載の方法。
- 前記沈殿微生物が超音波または機械的処理によって分解される、請求項1〜請求項11のいずれか1項に記載の方法。
- 前記沈殿微生物が蟻酸またはトリフルオロエタン酸などの酸およびアセトニトリルを含む溶液によって分解される、請求項1〜請求項11のいずれか1項に記載の方法。
- 質量分析試料支持プレート上で測定試料を調製する工程をさらに含み、前記調製する工程が、前記分解微生物の可溶性タンパク質が埋め込まれている結晶のマトリックス支援レーザー脱離/イオン化のためのマトリックス物質を使用して実施される、請求項12または請求項13に記載の方法。
- 測定試料を調製する工程をさらに含み、前記調製する工程が、マトリックス支援レーザー脱離/イオン化のためのマトリックス物質を備えた溶液を添加する質量分析試料支持体上に、前記沈殿物の微生物の一部を移すことを含む、請求項1〜請求項13のいずれか1項に記載の方法。
- 前記質量分析測定が、マトリックス支援レーザー脱離イオン化法(MALDI)によるイオン化を備えた飛行時間質量分析計で実施される、請求項14または請求項15に記載の方法。
- 前記溶液が5〜20%のドデシル硫酸ナトリウム(SDS)水溶液である、請求項5または請求項6に記載の方法。
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FR2942806B1 (fr) * | 2009-03-03 | 2011-09-02 | Assist Publ Hopitaux De Paris | Procede d'identification de germes en milieu liquide |
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DE102010023452B4 (de) | 2010-06-11 | 2012-11-08 | Bruker Daltonik Gmbh | Massenspektrometrische Messung von β-Lactamase-Resistenzen |
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US9551020B2 (en) * | 2011-04-21 | 2017-01-24 | Biomerieux, Inc. | Method of detecting at least one mechanism of resistance to carbapenems by mass spectrometry |
US9631221B1 (en) | 2011-09-02 | 2017-04-25 | Becton, Dickinson And Company | Identification of microorganisms using MALDI-TOF-MS on-plate extraction |
US9074236B2 (en) * | 2012-05-01 | 2015-07-07 | Oxoid Limited | Apparatus and methods for microbial identification by mass spectrometry |
DE102012011647B4 (de) * | 2012-06-08 | 2020-07-02 | Bruker Daltonik Gmbh | Analyse von Mikroben aus Mikrokolonien mittels MALDI-Massenspektrometrie |
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CA3225630A1 (en) | 2021-07-15 | 2023-01-19 | Jerome Lemoine | Identification of microorganisms based on identification of peptides using a liquid separation device coupled with a mass spectrometer and processing means |
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US4753749A (en) | 1984-03-08 | 1988-06-28 | Interface Research Corporation | Microbiocidal cleaning agent and preparation thereof |
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EP0453580A1 (en) | 1990-03-01 | 1991-10-30 | Shell Internationale Researchmaatschappij B.V. | Fungicidal benzyl-tris(aryl)-phosphonium salts |
US6177266B1 (en) * | 1996-04-29 | 2001-01-23 | The United States Of America As Represented By The Secretary Of The Army | Rapid identification of bacteria by mass spectrometry |
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AU2001288762A1 (en) | 2000-09-08 | 2002-03-22 | Large Scale Proteomics Corporation | Detection and characterization of microorganisms |
US6996472B2 (en) * | 2000-10-10 | 2006-02-07 | The United States Of America As Represented By The Department Of Health And Human Services | Drift compensation method for fingerprint spectra |
EP1556684A4 (en) * | 2002-11-01 | 2008-01-23 | Univ Colorado Regents | QUANTITATIVE ANALYSIS OF PROTEIN ISOFORMS USING FLIGHT TIME MASS SPECTROMETRY BY MATRIX ASSISTED LASER DESORPTION / IONIZATION |
WO2009015484A1 (en) † | 2007-08-02 | 2009-02-05 | Universite Laval | Concentration and enrichment of microbial cells and microbial nucleic acids from bodily fluids |
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US10059975B2 (en) * | 2008-10-31 | 2018-08-28 | Biomerieux, Inc. | Methods for the isolation and identification of microorganisms |
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