この作品は、付与RC3の​​AI089106の下で国立衛生研究所（NIH）によってサポートされていました。 多剤耐性結核核酸は、熱帯病研究訓練のための国連児童基金/国連開発計画/世界銀行/世界保健機関特別プログラム（TDR）、ジュネーブ、スイスから提供された。 我々は、プロトタイプアッセイに含める特定の遺伝子突然変異についてのガイダンスのために米国疾病対策予防センターの博士トムShinnickに感謝します。

ユニバーサルPCR法上の注意事項に従ってくださいラボラトリーズためには、基板ごとに複数の増幅マイクロアレイとガスケットを含み、ここで説明したように、バルクコンテナで同時にすべての増幅マイクロアレイを洗浄するために、運用が効率的です。他の場所で30,33報告されているように消耗品のフォーマットは、完全に密閉され、閉じられたアンプリコンテストで増幅後のマイクロアレイの洗浄工程を実行するために用意されています。 1。セットアップ<ol><li>抽出と選択の方法で適当な生物学的安全性条件下で試料から核酸を精製する。要件は、核酸は、非対称多重PCR増幅のために十分な純度であることである。 </li><li>汚染除去のソリューションと表面と機器を拭いて、ワークスペースを準備します。 </li><li>氷や冷たいブロックに増幅試薬を配置します。 </li><li>増幅マスターミックスのための無菌1.5mlマイクロチューブにラベルを付けて、滅菌した0.2ミリリットルマイクロ遠心TUにラベルを付ける各サンプルについても。 </li><li>試薬は、簡単に渦を解凍し、ミニ遠心機でのパルススピンでチューブの底に内容を収集した後。 Taqポリメラーゼをボルテックスしないでください。穏やかに混合し、ミニ遠心機でのパルススピンに従うようにチューブをフリック。 </li><li>可能なピペットの不正確さを考慮するために、表1に示すサンプルごとの反応容量を用いた増幅マスターミックスを調製し、処理中のサンプルの総数よりも少なくとももう1つの反応容積を調製する。何度muliplex PCRバッファーを備えているものの上に、マスターミックスは、追加のTaqポリメラーゼが含まれていることに注意してください。他のすべての試薬が組み合わされた後にマスターミックスを増幅/禁止制御を追加し、ストック·試薬チューブは、ストレージに戻った。 </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 試薬 </td><td> サンプルごとの容量（μL） </td><td> &lt;強い&gt;ファイナル[] </td></tr><tr><td>ホットスターのTaq PlusをMulitplex PCRバッファー</td><td> 25 </td><td> 1X </td></tr><tr><td>ウシ血清アルブミン（BSA） </td><td> 0.55 </td><td> 0.6 mg / mlの </td></tr><tr><td>ホルムアミド</td><td> 3.8 </td><td> 7.6％ </td></tr><tr><td>追加のTaqポリメラーゼ</td><td> 0.8 </td><td> ユニット/μl（4台の合計） </td></tr><tr><td> MDR-TBのプライマーミックス</td><td> 15.75 </td><td> - </td></tr><tr><td> RNaseフリーのH 2 O </td><td> 2.1 </td><td> - </td></tr><tr><td>増幅/禁止の制御</td><td> 1 </td><td> 5.0 FG /μL </td></tr><tr><td>合計</td><td> 49 </td><td></td></tr></tbody></table> 表1。MDR-TBの増幅マイクロアレイマスターミックス組成物。 <ol start="7"><li>渦マスターミックス、ミニ遠心機でのパルススピンでチューブの底に内容を収集します。 </li><li>マスターミックス49μLとMの1μLを組み合わせる上記工程1.4からの試料管の各々に結核 DNA。外部、鋳型なし対照（NTC）は、M.の代わりに、分子生物学グレードの水の1μlを使用する結核 DNAサンプル。渦とパルススピン仕上げチューブ（S）。 </li></ol> 2。増幅マイクロアレイをロード<ol><li>マイクロアレイ自体には手を触れないように注意して、それぞれのマイクロアレイの中心に、各マスターミックス/サンプルの48を添加する。 </li><li>マイクロアレイチャンバー内の気泡ではないトラップに気をつけながら、マイクロアレイガスケットおよびシールの上にカバースリップを配置。気泡が負に増幅効率に影響を与える可能性そして、その後のマイクロアレイ画像にアーチファクトやノイズを作成することがあります。 </li></ol> 3。熱サイクル<ol><li>いったんすべてのマイクロアレイは、フラットブロックサーマルサイクラー上のサンプル、場所基板が装填されています。加熱蓋オプションが「オフ」になっていることを確認してください。 Akonniバイオシステムズから入手した機器は、既に使用のために事前資格になります。それ以外の場合は、フラットブロックサーマルサイクラー（s）が（複数の）均一な、全てのマイクロアレイ基板で一貫した加熱を提供することを検証することは非常に重要である。 </li><li>サーマルサイクラーソフトウェアを開き、適切なプログラムを選択して、反応体積は、「50μL」と入力し、実行を開始します。ここで説明する熱サイクルプロトコルは、で構成されています。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 熱サイクル手順 </td></tr><tr><td> 1 </td><td> 88℃ </td><td> 5分</td></tr><tr><td> 2 </td><td> 88℃ </td><td> 30秒</td></tr><tr><td> 3 </td><td> 55℃ </td><td> 1分</td></tr><tr><td> 4 </td><td> 65℃ </td><td> 30秒</td></tr><tr><td> 5 </td><td> 50サイクルの工程（2-4）を繰り返す</td></tr><tr><td> 6 </td><td> 65℃ </td><td> 3分</td></tr><tr><td> 7 </td><td> 55℃ </td><td> 3時間</td></tr></tbody></table></li><li>必要に応じて、または特定の実験に応じて全熱サイクル数と55°Cの増幅後の保持時間は、ユーザが変更することができることを独立した変数である。 MDR-TBのマスターミックスは、非対称（主に一本鎖）アンプリコンを作成するので、それは他の点では従来、指数関数的なPCR増幅プロトコルのために利用されているより多くの熱サイクルを含めるようにして、通常は便利です。 </li><li>熱サイクルとハイブリダイゼーションのプログラムが完了したら、プログラムを終了増幅マイクロアレイを削除、ソフトウェアを終了し、サーマルサイクラー（複数可）をオフにしてください。</li></ol> 4。洗浄及び乾燥<ol><li>同時に24基材への洗浄のために、1×SSPE少なくとも250ミリリットル調製 - 0.1％トリトンX-100洗浄緩衝液、および脱イオン水またはそれぞれのMilli-Qグレード水は少なくとも250 mlの二つの容器を。洗浄緩衝液を予め調製することができる。 </li><li>慎重にフラットエンドピンセットを用いて、各増幅マイクロアレイ室からカバーガラスとガスケットを取り外します。 この手順では、物理的に損傷ゲル素子アレイの最大の危険性がある点である。増幅された核酸の拡散を防止するための適切なPCR汚染コントロールを使用して、作業環境の中。 </li><li>組織学のスライドホルダーにマイクロアレイ基板を配置し、洗浄バッファーを含む洗浄ビンにすべてのスライドを配置。蓋付きの容器をカバーしています。 </li><li>穏やかに攪拌しながら室温で10分間、マイクロアレイを洗浄する。 </li><li>脱イオンの2連続洗浄にマイクロアレイを3回ずつ浸したりミリQグレードの水、そして空気乾燥。 </li></ol> 5。イメージング非対称のMDR-TBのプライマーミックスは、Cy3標識アンプリコンを生成します。ゲル素子マイクロアレイのCy3を撮像することが可能な任意の標準的マイクロアレイイメージャーで画像化することができる。次の撮像手順はAkonniが提供する多剤耐性結核マスターミックス、Dx2100フィールドポータブルイメージャ、および自動化された解析ソフトウェアに特異的である。 <ol><li>イメージャからのイーサネットケーブルがコンピュータに接続されていることを確認します。 </li><li>イメージャの電源をオンにします。電源スイッチが機器の背面パネルに配置されている。イメージャの電源を入れたときのイメージャの前面にある青いLEDが点灯します。 </li><li>コンピュータの電源を入れます。 </li><li>コンピュータのデスクトップ上で、自動化された画像解析ソフトウェアを起動するためのソフトウェアのアイコンをダブルクリックします。 </li><li>イメージャカメラとの接続を確立するためのソフトウェアのを待ちます。接続が確立されると、分析ボタンが使用可能になり、メッセージ「接続成功」とは、画面の左下隅に表示されます。 </li><li>マイクロアレイは、ユーザから、対物レンズに向かって反対側で乾燥させた増幅マイクロアレイを挿入します。マイクロアレイが不適切レンズとカメラに対して配向されている場合は、自動化された分析ソフトウェアは、蛍光画像上にグリッドを配置するために失敗する。 </li><li>アクセスドアを閉じます。プレビュー画像は、コンピュータの画面上で利用できるようになります。 </li><li>ドロップダウンメニューから多剤耐性結核解析スクリプトを選択し、（ミリ秒単位）所望の露光時間を入力します。ゲル素子増幅マイクロアレイのための典型的な出発点は、100〜500ミリ秒である。 </li><li>飽和画素は赤でプレビュー画像に表示されます。個々のスポット内の飽和画素の数を最小化するために露光時間を調整する。 </li><li>蛍光画像を取得して分析する分析をクリックしてください。ソフトウェアが自動的に配置されますMDR-TBアッセイ特異グリッド画像上に、積分強度及びバックグラウンド値を抽出し、薬剤耐性及び遺伝子型決定の結果を報告する。自動化された分析では、典型的には数秒かかります。傷、埃、蛍光アーティファクト、またはマイクロアレイ機能への物理的な損傷が含まれている挑戦的なイメージの場合、ソフトウェアは分析を完了するために1分までが必要な場合があります。 </li><li>分析が完了したら、[保存]をクリックインストール先のフォルダを選択し、ファイル名を入力し、[OK]をクリックします。基礎となるマイクロアレイデータは。xlsファイルとして保存され、オフライン分析のために、必要に応じて、エクスポートすることができます。 </li><li>分析が完了すると、イメージャからの増幅マイクロアレイを削除し、次の分析を開始するために新規をクリックしてください。 </li><li>データの収集が完了したら、コンピュータをシャットダウンし、ソフトウェアを終了し、イメージャの電源を切ります。 </li></ol>

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Methods. 65, 294-300 (2006).</li><li>Brossier, F., Veziris, N., Truffot-Pernot, C., Jarlier, V., Sougakoff, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+the+genotype+MTBDR+line+probe+assay+for+detection+of+resistance+to+rifampin+and+isoniazid+in+strains+of+Mycobacterium+tuberculosis+with+low-+and+high-level+resistance.">Performance of the genotype MTBDR line probe assay for detection of resistance to rifampin and isoniazid in strains of Mycobacterium tuberculosis with low- and high-level resistance.</a> J. Clin. Microbiol. 44, 3659-3664 (2006).</li><li>Padilla, E., Gonzalez, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+the+new+version+of+the+INNO-LiPA+Mycobacteria+and+GenoType+Mycobacterium+assays+for+identification+of+Mycobacterium+species+from+MB/BacT+liquid+cultures+artificially+inoculated+with+mycobacterial+strains.">Comparative evaluation of the new version of the INNO-LiPA Mycobacteria and GenoType Mycobacterium assays for identification of Mycobacterium species from MB/BacT liquid cultures artificially inoculated with mycobacterial strains.</a> J. Clin. Microbiol. 42, 3083-3088 (2004).</li><li>Barnard, M., Gey van Pittius, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diagnostic+performance+of+the+GenoType+MTBDRplus+Version+2+line+probe+assay+is+equivalent+to+that+of+the+Xpert+MTB/RIF+assay.">The diagnostic performance of the GenoType MTBDRplus Version 2 line probe assay is equivalent to that of the Xpert MTB/RIF assay.</a> J. Clin. Microbiol. 50, 3712-3716 (2012).</li><li>Sekiguchi, J. -. I., Nakamura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+a+line+probe+assay+for+rapid+identification+of+pncA+mutations+in+pyrazinamide-resistant+Mycobacterium+tuberculosis+strains.">Development and evaluation of a line probe assay for rapid identification of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis strains.</a> J. Clin. Microbiol. 45, 2802-2807 (2007).</li><li>Chandler, D. P., Bryant, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+amplification+microarrays+for+infectious+disease+diagnostics.">Integrated amplification microarrays for infectious disease diagnostics.</a> Microarrays. 1, 107-124 (2012).</li><li>Vincent, V., Rigouts, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TDR+Tuberculosis+Strain+Bank:+a+resource+for+basic+science,+tool+development+and+diagnostic+services.">The TDR Tuberculosis Strain Bank: a resource for basic science, tool development and diagnostic services.</a> Int. J. Tuberc. Lung Dis. 16, 24-31 (2012).</li><li>Golova, J. B., Chernov, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-volatile+copolymer+compositions+for+fabricating+gel+element+microarrays.">Non-volatile copolymer compositions for fabricating gel element microarrays.</a> Anal. Biochem. 421, 526-533 (2012).</li><li>Cooney, C. G., Sipes, D., Thakore, N., Holmberg, R., Belgrader, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plastic,+disposable+microfluidic+flow+cell+for+coupled+on-chip+PCR+and+microarray+detection+of+infectious+agents.">A plastic, disposable microfluidic flow cell for coupled on-chip PCR and microarray detection of infectious agents.</a> Biomed. Microdevices. 14, 45-53 (2012).</li><li>Lane, S., Evermann, J., Loge, F., Call, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplicon+secondary+structure+prevents+target+hybridization+to+oligonucleotide+microarrays.">Amplicon secondary structure prevents target hybridization to oligonucleotide microarrays.</a> Biosens. Bioelectron. 20, 728-735 (2004).</li><li>El-Hajj, H. H., Marras, S. A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+sloppy+molecular+beacon+probes+for+identification+of+Mycobacterial+species.">Use of sloppy molecular beacon probes for identification of Mycobacterial species.</a> J. Clin. Microbiol. 47, 1190-1198 (2009).</li><li>Boehme, C. C., Nabeta, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+molecular+detection+of+tuberculosis+and+rifampin+resistance.">Rapid molecular detection of tuberculosis and rifampin resistance.</a> N. Engl. J. Med. 363, 1005-1015 (2010).</li></ol>

著者らは、競合する経済的利益を宣言していません。

増幅マイクロアレイとの多重化の程度は、最終的には、マルチプレックス非対称PCRの効率ではなく、マイクロアレイによって決定される。我々の経験では、一意の10〜12のプライマー対を容易に増幅マイクロアレイフォーマットで多重化することができる。従来のプライマーおよびプローブの設計基準のでその一つはこれもPCR緩衝液中の溶液相核酸および固定化されたマイクロアレイプローブ...

早期患者発見と迅速な治療が結核菌を減らす最も効果的な制御戦略（MTB）変速機1と見なされ、ポイントオブケア（POC）またはPOCテストの近くには、同時に結核を診断することを結核コミュニティの幅広い合意が今そこにあるアール薬剤耐性（DR）が必要である。例えばCepheid社のGeneXpertおよび他の核酸増幅試験などの技術は、多くのTB患者のための診断までの時間を短縮し、リファンピンまたは他の第一または第二選択薬2に対する耐性を付与する選択された変異に耐性を示す急速な読み出しを提供する。リアルタイムおよび等温核酸増幅試験は、MDR-TBをもたらす薬剤耐性突然変異を同定するために設計されているが、それらは検出突然変異のスペクトルは、患者の薬剤耐性プロファイルに対応する個別化された薬物療法を設計することがしばしば不十分であるおよび関連する技術的な制約光クロストークまたは増幅および報告化学の複雑さのために3-7座位または検出された変異の数を制限することがあります。したがって、より高い多重化容量を有する検出技術は、MDR-TBのPOC診断において公知のギャップに対処するために必要とされる。 マイクロアレイとWHOが承認しハインラインプローブアッセイは、MDR-TBの8月29日を診断する「複数の遺伝子、複数の変異「課題に対処することができます。残念ながら、これらのハイブリダイゼーションに基づく、多重化された検出プラットフォームは、多段階で複雑、かつ分子技術の大幅な訓練と技能を必要とするオープンアンプリコンプロトコルを使用しています。増幅マイクロアレイ30は 、これらのマイクロアレイワークフローと運用の問題の一部に対処するために設計されました。簡略化流体原理は、増幅ハイブリダイズし、単一のマイクロ流体チャンバ内での核酸標的を検出することである。ユーザーは、核酸増幅MASを紹介TERはピペットで流体室に混ぜて熱サイクルプロトコルを開始します。ここに示されているバッチ処理法では、マイクロアレイは、その後、バルク溶液中で洗浄し、乾燥させ、結像される。この研究は、 のrpoBのためのMDR-TBのマイクロアレイ試験（30の変異）、 のkatG（2の変異）、 仁荷 （4の変異）、 のrpsL（2の変異）、embB（1変異）、IS1245、IS6110を用いた増幅マイクロアレイの機能性を実証、および内部増幅およびハイブリダイゼーションコントロール。マイクロアレイプローブ（野生型（WT）及び一塩基変異体（MU））の少なくとも一つのマッチしたペアは、関心のある各変異のために含まれている。多剤耐性Mから精製された核酸結核は 、TDR結核菌株バンク31からのものである。他の箇所32記載のようにゲル素子マイクロアレイは、我々がpolymeriz各プローブ4％のモノマーおよび0.05mMを使用することを除いて、実質的に共重合させることによりガラス基板上に製造されるATION混合物。アレイは、使用前の50mlガスケットで囲まれている。熱サイクル、ハイブリダイゼーションおよび洗浄工程の後、増幅マイクロアレイはAkonni携帯型分析に結像される。バックグラウンド補正、統合された信号強度は、生から得られる。tifの画像を、固定円アルゴリズムを使用して。各ゲル素子のノイズは、ローカルスポットのバックグラウンドの標準偏差の3倍として計算される。対雑音比（SNR）、信号用の≥3が値遺伝子標的は、典型的には、検出と見なされる。各遺伝子または特定のコドンにおける変異の有無を決定するために、判別比は、（WT-MU）/（WT + MU）とし​​てSNR値から計算される。 0野生型配列の指標である判別比&lt;比は、一方0は、遺伝子座における薬剤耐性変異の指標である&gt;は。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;">MDR-TB amplification microarrays, with applied gasket and pre-cut cover slips</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Multiplex PCR kit, containing 2X PCR buffer with HotStar Taq plus</td>
			<td>Qiagen</td>
			<td>#206143</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Taq polymerase</td>
			<td>Qiagen</td>
			<td>#201207</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">RNAse-free water</td>
			<td>Qiagen</td>
			<td>#206143&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Formamide</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP227-500</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">20 mg mL-1 non-acetlyated bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>#3B6917</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">5X concentrated MDR-TB primer mix</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">500 fg uL-1 amplification and inhibition control</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">20X SSPE</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP1328-4</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Triton X-100</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP151-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Disinfecting Spray&#160;</td>
			<td>Current Technologies, Inc.</td>
			<td>#BRSPRAY128</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">70% Isopropyl Alcohol</td>
			<td>Decon Labs, Inc.</td>
			<td>#8416</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Equipment</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Microarray imager, with automated image and data analysis software</td>
			<td>Akonni Biosystems</td>
			<td>100-20011</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Thermal cycler with flat block insert</td>
			<td>Akonni Biosystems</td>
			<td>100-10021</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">High-throughput wash station and slide holder</td>
			<td>ArrayIt</td>
			<td>HTW</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dissecting forceps</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#10-300</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Mini Vortexer</td>
			<td>VWR</td>
			<td>#3365040</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Mini-centrifuge</td>
			<td>VWR</td>
			<td>#93000-196</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Airbrush Compressor Kit</td>
			<td>Central Pneumatic</td>
			<td>#95630</td>
			<td></td>
		</tr>
	</tbody>
</table>

demonstrating a multi-drug resistant mycobacterium tuberculosis amplification microarray

マイクロアレイワークフローを簡略化する日常的低リソース環境において使用することができるMDR-TBマイクロアレイベースの診断を作成するために必要な最初のステップである。増幅マイクロアレイは単一の溶液中で、単一のマイクロ流体チャンバ内に非対称PCR増幅、目標サイズの選択、標的標識、およびマイクロアレイハイブリダイゼーションを組み合わせる。バッチ処理法は、9 -プレックス非対称マスターミックス及び多剤耐性結核菌 （MDR-TB）を遺伝子型決定するための低濃度のゲル素子マイクロアレイを用いて実証されている。ここで説明するプロトコルは、6時間で完了し、ゲノムDNAの少なくとも1,000細胞同等で正しい遺伝子型決定を提供することができる。オンチップの洗浄工程を組み込むことは完全に閉じアンプリコンの方法およびシステムになるであろう、実現可能である。増幅マイクロアレイでの多重化の程度は、最終的に単一のマスタmに組み合わせることができるプライマー対の数によって制約される九依然としてむしろアレイ上に固定化されるプローブの数よりも所望の感度および特異性のパフォーマンス·メトリックを達成する。同様に、総分析時間は、特定の使用目的に、研究課題、および検出の所望の限度に応じて短縮または延長することができる。それにもかかわらず、一般的なアプローチは、有意に手動集約的で時間のかかる処理工程の数を減らすことによって、エンドユーザーのためのマイクロアレイのワークフローを効率化し、日常的な臨床実践にマイクロアレイに基づく診断を翻訳するための簡略化された生化学的およびマイクロ流体経路を提供する。

定性的画像分析は、自動画像解析ソフトウェアにより生成されたデータテーブルに識別するために挑戦している実験的なノイズやばらつきの原因への洞察を提供することができる。このように、それは1）すべてのゲルの要素が無傷で損傷がない、2）グローバルなバックグラウンドノイズ比（SNR）の値に個々の信号に影響を与える可能性のある蛍光成果物がないことを確認するため、視覚的?...

Watch this Scientific Journal Video about Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray at JoVE.com

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

増幅マイクロアレイは大幅にエンドユーザーのために、マイクロアレイのワークフローを合理化し、単一の室内に非対称PCR増幅およびマイクロアレイハイブリダイゼーションを兼ね備えています。マイクロアレイワークフローを簡略化する日常的低リソース環境において使用することができるマイクロアレイに基づく診断を作成するために必要な最初のステップである。

多剤耐性を実証<em&gt;結核菌</em&gt;増幅マイクロアレイ

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

demonstrating-multi-drug-resistant-mycobacterium-tuberculosis

Research

JoVE Journal

Immunology and Infection

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

12.6K ビュー.  Akonni Biosystems, Inc.. 増幅マイクロアレイは大幅にエンドユーザーのために、マイクロアレイのワークフローを合理化し、単一の室内に非対称PCR増幅およびマイクロアレイハイブリダイゼーションを兼ね備えています。マイクロアレイワークフローを簡略化する日常的低リソース環境において使用することができるマイクロアレイに基づく診断を作成するために必要な最初の?...

ビデオ: Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

マイクロタイタープレート形式で液体希釈法により、第一、第二ラインの薬のための結核菌の複合体の抗菌薬感受性試験

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

免疫・感染学

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

のサンプル調製<em&gt;結核菌</em核磁気共鳴代謝研究のための&gt;抽出

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

の成長結核バイオフィルム

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

自然送信時に結核マラリア重複感染を研究する実験モデル結核菌とマラリアベルゲイ</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

回復への新規マイクロダイセクションアプローチ<em&gt;結核菌</em肺肉芽腫、ホルマリン固定パラフィン包埋から&gt;特異的転写

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

上の機能的研究のための3Dヒト肺組織モデル<em&gt;結核菌</em&gt;感染

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

細胞内の標的阻害剤の有効性及び細胞毒性スクリーニングのためのシステム<em&gt;結核菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

マクロファージ継代に対する薬効を評価するために、ハイスループットアッセイの互換性<em&gt;結核菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

18FDG PET/CT イメージング勉強結核感染と非ひと霊長類における治療のためのツールとしての解析

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

レポーター酵素蛍光を用いたマウスにおける結核菌をイメージング

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...