我々は、結核の成長タイムラプスビデオセグメントに対してショーンフィッツウォーターとカルメンGianninaルナコロンボに感謝します。我々は、現在のプロトコルは主に逐語的に取得されたユーザガイドを、編集中に彼女の徹底と優れたフィードバックのためマーティローパーを感謝し、共同編集。このビデオの制作は、NIH /フォガティ国際センターによって資金を供給された<a href="http://www.fic.nih.gov/">http://www.fic.nih.gov/</a>デビッドAJムーアが、ロンドン大学インペリアルカレッジで感染症の熱帯医学とリーダーのウェルカムトラスト臨床研究員（フェローとして貢献受賞番号078067/Z/05）。マークF. BradyはNIH /フォガティ国際センターの研究員として貢献した。

<ol><li> ストック溶液を準備する <ol><li>リン酸塩緩衝在庫<ol><li>一塩基性リン酸カリウム溶液（千ミリリットル蒸留水に溶解したリン酸カリウム一塩基の9.07グラム）の950ミリリットルでナトリウム塩基性溶液（千ミリリットルの蒸留水に溶解したナトリウム二塩基性リン酸の9.47グラム）の950ミリリットルを混合し、かき混ぜ、調整して戻ってそれぞれのソリューション50mlの維持pHは必要に応じて</li><li> 6.8 ± 0.2にpHを調整する：pHを上昇させるために追加のリン酸ナトリウム二塩基性溶液、低いpHに一塩基性リン酸カリウム溶液を入れる</li><li> 121から124まででオートクレーブ℃で15分間の滅菌に</li><li>プレート無菌性を確認するために48時間で37ペトリ皿とインキュベートの栄養寒天培地上にリン酸緩衝液° C100μlのアリコートを</li><li> 2〜8℃冷蔵庫で保管℃で最長1ヶ月まで 注 ：各痰のサンプルでは、リン酸緩衝液10mlのが必要です </li></ol></li><li>除染株式<ol><li>水酸化ナトリウム溶液（200ミリリットル滅菌蒸留水に溶解した水酸化ナトリウムの8.0グラム）とクエン酸ナトリウム溶液（200ミリリットル滅菌蒸留水で蒸留クエン酸ナトリウムの5.8グラム）の等容量を組み合わせる</li><li> 15分間121〜124℃で混合し、オートクレーブ</li><li> 2〜8℃冷蔵庫で保管℃で最長1ヶ月まで 注 ：各痰のサンプルは、汚染除去の株式の2ミリリットルが必要です </li></ol></li><li>文化メディア株式<ol><li>滅菌蒸留水900ミリリットル中にグリセロールと7H9培地の粉末のcasitoneと5.9グラムの1.25グラムの3.1ミリリットル加える。一定に撹拌しながら混合を完全に溶解するまで</li><li> 15分間121〜124℃でオートクレーブ</li><li>滅菌サンプル調製のための16倍、100mmのガラス管、および抗生物質のソリューションと内部統制の準備のための10.8ミリリットルアリコートで4.5ミリリットルのアリコートに無菌培地を冷却し、分割</li><li> 37℃48時間の不妊（濁度の欠如）を確認する</li><li> 2〜8℃キャップ付きで保存するには、しっかりと一ヶ月にするため、最大閉鎖 注 ：各痰のサンプルでは、7H9培地のいずれかのチューブを含む4.5ミリリットルが必要です </li></ol></li><li>抗生物質ストック溶液<ol><li> A.イソニアジド株式（8 mg / ml）を<ol><li> 2.5ミリリットル滅菌蒸留水で完全にイソニアジド20mgを溶かす</li><li>水性溶媒用0.2μmのシリンジフィルターでフィルタリング</li><li> 6ヵ月まで-20℃で滅菌マイクロ遠心チューブに20μlのアリコートの店 注 ：各ストアド20μlの分画は、最大100サンプル（無駄を含む）のために十分である </li></ol></li><li> B.リファンピシン株式（8 mg / ml）を</li></ol><ol><li> 1.25ミリリットルのDMSOに完全に20mgをリファンピシンを溶かす</li><li> 1.25ミリリットル滅菌蒸留水とミックスを追加する</li><li>有機溶剤用0.2μmのシリンジフィルターとフィルター</li><li> 6ヵ月まで-20℃で滅菌した微量遠心チューブに20μlのアリコートの店 注 ：各ストアド20μlの分画は、最大100サンプル（無駄を含む）のために十分である </li></ol></li></ol></li><li> ワーキング溶液の調製法 <ol><li>汚染除去作業ソリューション<ol><li>必要な除染液の各20ミリリットルで、NALC結晶の0.1グラムを溶かし 注 ：各サンプルには、除染液の2ミリリットル必要があります 注意 ：NALCは時間が経過すると、その粘液溶解活性を失うように24時間後に未使用の状態にある任意のNaOH - NALCの除染液を捨てる </li></ol></li><li>文化メディアワーキングソリューション<ol><li>着手： <ol><li>処理されるすべての喀痰サンプルの7H9培地1チューブ、加えて全てのプレートに1追加のチューブ（ネガティブコントロールの列のため） </li><li>ポジティブコントロールの7H9培地の2チューブ（3 monoresistant菌株が使用されている場合） </li><li>抗生物質溶液の調製のための10.8ミリリットル7H9で1〜チューブ</li></ol></li><li>各サンプルチューブに7H9の4.5ミリリットルに0.5ミリリットルOADCを追加</li><li> 10.8ミリリットル7H9でチューブに1.2ミリリットルOADCを追加</li><li>抗生物質溶液の調製に使用される12ミリリットル7H9 - OADCで5ミリリットルポジティブコントロール用の7H9 - OADC、及びチューブ（S）で2チューブを脇に置いて（これらは、PANTAを必要としない） </li><li>再構成のPANTAと各サンプルチューブに、陰性対照のチューブ（7H9 - OADC - PANTA：総量= 5.1ミリリットル）に0.1ミリリットル追加 注：PANTA（7H9 - OADC - PANTA）との完全な培地は喀痰サンプルとネガティブコントロールに使用されている。ポジティブコントロールのためにと抗生物質溶液の調製のためのPANTAなく7H9 - OADCを使用する </li></ol></li><li>抗生物質ワーキングソリューション<ol><li> 1。未使用の24ウェルプレートでの抗生物質ワーキングソリューションを作成する手順については、図1を参照してください 注 ： 正確な感受性の結果を得るには、最終的な抗生物質の濃度が重要である。次の手順では、lに適していますマイクロピペットを装備aboratories。マイクロピペットが使用できない場合は、ツベルクリン注射器はmodsperu.orgでユーザガイド付録3で説明されている希釈の異なるシリーズで使用できます 注 ： 薬物活性として再凍結または再使用しないで抗生物質の作業または原液が失われる可能性があります処理の日の図1の終了時にす ​​べての未使用の抗生物質を破棄。。抗生物質ワーキングソリューションを作成（modsperu.orgにおけるユーザガイドから） </li></ol></li></ol></li><li> 痰のサンプルおよびプレートの準備 <ol><li>汚染除去<ol><li> 15ミリリットルの遠心チューブに痰のサンプルの場所の2ミリリットル（小さい場合、リン酸緩衝液で2ミリリットルに構成している、多くの場合、唯一の2ミリリットル使用） </li><li> 2ミリリットルをNaOH - NALCのソリューションを追加します。 </li><li> 20秒のためのキャップをしっかりと管と渦、水酸化ナトリウム、NALCのソリューションの連絡先を内部全体の管の表面と蓋を確実にするためにチューブを反転させる</li><li> 15分以上放置する - サンプルは、特に粘性の場合は数分で延長することができます。治療上のを避けるために、20分を超えないようにしてください </li><li> 4倍をアルカリ中和し、除染プロセスを終了し、チューブを反転させてよく混合してリン酸緩衝液（pH6.8）で14ミリリットルにチューブを埋める</li><li> 15分間、3000gで遠心（3000 gを達成するために必要な分当たりの回転の方程式のためmodsperu.orgでユーザーガイドの付録2を参照） </li><li>慎重に10％の次亜塩素酸ナトリウムまたは他の適切な消毒剤と液体廃棄物の容器に上清を取り除き、ペレットを保持する</li></ol></li><li>最終サンプルのサスペンションとバックアップの準備<ol><li> 7H9 - OADC - PANTAを（5.1ミリリットルを含むチューブから）を使用して、パスツールピペットで遠心管に2ミリリットルの合計体積に試料ペレットを再懸濁します。うまくミックス</li><li>バックアップとして2〜8サンプルの懸濁液とマイクロ遠心チューブ内の店舗° Cの1ミリリットルを削除します。 </li><li>残りの7H9 - OADC - PANTAとチューブにサンプルのサスペンションの第1ミリリットルを追加、よく混ぜる。これはメッキの準備ができて最終的なサンプルのソリューションです。 </li></ol></li><li> 24ウェルプレートに試料懸濁液の配置<ol><li> 24ウェルプレート内の列の4ウェルの各々に最終的なサンプルの懸濁液900μl場所</li><li> 、列3を除いて、プレートのすべての列まで、追加のサンプルを繰り返して（またはすべてのサンプルがメッキされるまで）満ちている</li><li>各サンプルプレートの列3（負の内部統制）の4ウェルにサンプルなし 7H9 - OADC培地の900μl場所</li></ol></li><li>サンプルに抗生物質を追加する<ol><li>マルチチャンネルピペットを使用して、慎重に7H9 - OADCと抗生物質ワーキングソリューション（抗生物質希釈で2列目は、プレート - 参照図1）でウェルから100μlの4のヒントを埋める</li><li> プレートやサンプルに触れることなく、900μlのサンプル溶液と1列目のウェルに100μlのアリコートを追加</li><li>すべての列は、媒体の追加の100μlの（薬物のない井戸）または（ネガティブコントロールのカラム3を含む）の抗生物質を受信するまで繰り返します。 </li><li>密閉式ポリエチレン（ジップロック）袋とシールで、その蓋と場所でプレートを閉じて（ 袋はこの時点から再び開いていない ） </li><li> 37℃（CO 2濃縮は不要です） </li></ol></li></ol></li><li> 品質管理 <ol><li> ネガティブコントロールは、各プレート上で実行されています。井戸のいずれかで、成長の存在は、ネガティブコントロールの列で、プレート全体が廃棄とサンプルの再メッキする必要があるため、クロスコンタミネーションを示します。 </li><li> ポジティブコントロールは、一日の終わりに別々のプレート上で実行されています。One完全に薬剤感受性株の列で実行され、INHとRIFに歪み耐性が別の列で実行されている。 MDR菌株を実行している懸念がある場合は、INH monoresistantひずみとRIF monoresistant歪みの両方が影響を受けやすいとMDR株の代わりに実行することができます。すべてのポジティブコントロールには、そのメディアのサポートの成長を実証するために薬物のない井戸の成長が必要です。薬物濃度が正しいことを示すために、感受性株はINHまたはRIF井戸に成長しないはずですが、MDR株はINHとRIF井戸にも拡大成長してください。 </li></ol></li><li> プレートの読み取り <ol><li>としてめっきの日に&quot;0日目、&quot;板の読み込みは5日目に開始し、負の場合は目に5測定値は、14日目とその後15日目から毎年2〜3日まで（またはワークロードに応じて別の日）は毎日行われます。 -21。プレートを上検討されています10倍の目標と倒立光学顕微鏡</li><li> 肯定的な文化は、薬物のない井戸の両方で複数のコロニー形成単位は（&gt; 2 CFU）が存在する環境です。初期のマイコバクテリアの成長は、小さな湾曲したコンマまたはらせん（日5-9）のように見えます。コロニー形成は、通常、&quot;コード&quot;または&quot;もつれ&quot;（modsperu.org上のイメージのライブラリを参照してください）​​へと進行する</li><li>日には文化が2薬物のない井戸で陽性である、薬物含有ウェルを読んでおく必要があります。 薬剤の存在下で任意の成長がその薬剤に耐性を示す。 注 ： 正の培養は薬物のない井戸に記載されている&gt; 1週間後に薬物含有井戸の読み取りが行われる場合、この、成長のブレークスルーとしての抵抗を表していない可能性がありますが時折見られます </li><li> 21日目で陽性ではないサンプルは負の文化と考えられている</li><li>井戸の濁りは、マイコバクテリアの成長は、曇りが発生しないのに対し、汚染の増殖の結果である</li><li> 21日間培養の終了時に破棄されることがあります。文化は、彼らの密封袋に残り、オートクレーブバッグに入れて45〜60分間121〜124℃でオートクレーブしている</li></ol></li></ol>

<ol>
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E. <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+microscopic+observation+drug+susceptibility+assay+in+drug+susceptibility+testing+for+Mycobacterium+tuberculosis.">Performance of the microscopic observation drug susceptibility assay in drug susceptibility testing for Mycobacterium tuberculosis.</a> J Clin Microbiol. 40, 4750-4750 (2002).</li><li>Shiferaw, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+microscopic+observation+drug+susceptibility+assay+for+detection+of+multidrug-resistant+Mycobacterium+tuberculosis.">Evaluation of microscopic observation drug susceptibility assay for detection of multidrug-resistant Mycobacterium tuberculosis.</a> J Clin Microbiol. 45, 1093-1093 (2007).</li><li>Tovar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+diagnosis+of+pleural+tuberculosis+using+the+microscopic-+observation+drug-susceptibility+technique.">Improved diagnosis of pleural tuberculosis using the microscopic- observation drug-susceptibility technique.</a> Clin Infect Dis. 46, 909-909 (2008).</li><li>Vargas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+sputum-scarce+HIV-associated+pulmonary+tuberculosis+in+Lima,+Peru.">Diagnosis of sputum-scarce HIV-associated pulmonary tuberculosis in Lima, Peru.</a> Lancet. 365, 150-150 (2005).</li></ol>

MODSのアッセイは、資源が限られた設定で対象としています。初めて、MODSは、結核の迅速な液体培養の検出のための能力をもたらし、テストあたりのすぐ下に$ 3で資源が限られた設定に耐性結核を多剤。 MODSは、非独占的、反復的な方法論であり、MODSのコミュニティは、常に他の研究室を作るために管理されていることの改善に興味を持っています。 液体がこぼれたり、エアロゾル化することができ?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Refrigerator/ freezer</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to store pre-prepared broth and antibiotic stocks</td>
</tr>
<tr>
<td>Vortex</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to aid sputum decontamination</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for sputum concentration; capable of reaching 3000 g; does not need to be refrigerated, but MUST be biosafe (buckets can be sealed)</td>
</tr>
<tr>
<td>Incubator (37 degree C)</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for culture; need not be CO2 enriched</td>
</tr>
<tr>
<td>Inverted light microscope</td>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to read MODS plates</td>
</tr>
<tr>
<td>Autoclave</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to sterilize media, PBS and used plates</td>
</tr>
<tr>
<td>Balance</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to weigh isoniazid, rifampicin and NALC</td>
</tr>
<tr>
<td>Middlebrook 7H9 broth (Difco)</td>
<td>Reagent</td>
<td>Fisher</td>
<td>DF0713-17-9</td>
<td>500gr/bottle; culture media base</td>
</tr>
<tr>
<td>Casitone (pancreatic digest casein)</td>
<td>Reagent</td>
<td>Fisher</td>
<td>DF0259&#x2010;17&#x2010;9</td>
<td>500gr/bottle; culture media base</td>
</tr>
<tr>
<td>Glycerol (glycerin) lyophilized</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G&#x2010;33&#x2010;500</td>
<td>500ml/bottle; culture media base</td>
</tr>
<tr>
<td>PANTA (Antibiotic mixture lyophilized BD) </td>
<td>Reagent</td>
<td>Fisher</td>
<td>B4345114</td>
<td>6 bottles/pack; antibiotic media supplement</td>
</tr>
<tr>
<td>OADC (Middlebrook OADC enrichment BD)</td>
<td>Reagent</td>
<td>Fisher</td>
<td>B11886</td>
<td>10 x 20ml/pack; nutritional media supplement</td>
</tr>
<tr>
<td>Dimethyl sulphoxide (Hibri-Max)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D-2650</td>
<td>100ml/bottle; to prepare rifampicin stock</td>
</tr>
<tr>
<td>Antibiotic stocks: isoniazid</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I-3377</td>
<td>50gr/bottle; direct susceptibility testing</td>
</tr>
<tr>
<td>Antibiotic stocks: rifampicin</td>
<td>Reagent</td>
<td>Sigma 99</td>
<td>R-3501</td>
<td>1gr/bottle; direct susceptibility testing</td>
</tr>
<tr>
<td>Sodium hydroxide (pellets)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>221465</td>
<td>500gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>Sodium citrate (trisodium salt dihydrate)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S-4641</td>
<td>500gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>N-acetyl-L-cysteine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-7250</td>
<td>50gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>Potassium Phosphate Monobasic crystal. KH2PO4</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P0662</td>
<td>500gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>Sodium Phosphate Dibasic, anhydrous. Na2HPO4</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0876</td>
<td>500gr/bottle; sputum decontamination</td>
</tr>
<tr>
<td>Sodium hypochlorite</td>
<td>Reagent</td>
<td>household bleach</td>
<td>&nbsp;</td>
<td>to discard contaminated waste</td>
</tr>
<tr>
<td>15ml centrifuge tubes (polypropylene 15ml Falcon 35&#x2010;2096)</td>
<td>Consumable</td>
<td>Fisher</td>
<td>14&#x2010;959&#x2010;49B</td>
<td>500ea/case; for sputum decontamination and concentration</td>
</tr>
<tr>
<td>24 well plates (Plates Tissue 24 wells BD Falcon 35&#x2010;3047)</td>
<td>Consumable</td>
<td>Fisher</td>
<td>08-772-1</td>
<td>50 plates/case; for culture and reading</td>
</tr>
<tr>
<td>Sealable polythene bags 6 X 6 &#x201C; (ziplock)</td>
<td>Consumable</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for biosecurity to contain 24 well plate</td>
</tr>
<tr>
<td>Glass tubes with lid (16 x 100mm and 18 x 145mm)</td>
<td>Consumable</td>
<td>VWR</td>
<td>47729-583</td>
<td>500 tubes/case; to store aliquots of prepared broth</td>
</tr>
<tr>
<td>Screw cap microcentrifuge tubes (1.5ml)</td>
<td>Consumable</td>
<td>Fisher</td>
<td>05&#x2010;669&#x2010;22</td>
<td>1000ea/case; to store aliquots of antibiotic stocks</td>
</tr>
<tr>
<td>0.22&#x03BC;m filters (aqueous solvents) Syringe filter Millex blue</td>
<td>Consumable</td>
<td>Fisher</td>
<td>SLGL 025 OS</td>
<td>50 units/case; to filter antibiotic stocks</td>
</tr>
<tr>
<td>0.22&#x03BC;m filters (organic solvents) Syringe filter Millex yellow</td>
<td>Consumable</td>
<td>Fisher</td>
<td>SLGV 033 RS</td>
<td>50 units/case; to filter antibiotic stocks</td>
</tr>
<tr>
<td>Disposable Pasteur pipettes borosilicate glass 9&#x2033;</td>
<td>Consumable</td>
<td>Fisher</td>
<td>13&#x2010;678&#x2010;20C</td>
<td>720ea/case; to mix PANTA with media mix</td>
</tr>
<tr>
<td>Aerosol barrier tips 1000&#x2010;1300&#x03BC;l</td>
<td>Consumable</td>
<td>Fisher</td>
<td>02&#x2010;707&#x2010;51</td>
<td>1000ea/pk; to dispense media into plate</td>
</tr>
<tr>
<td>USA Scientific Tips One 1&#x2010;200&#x03BC;l yellow tips</td>
<td>Consumable</td>
<td>Fisher</td>
<td>1111&#x2010;0006</td>
<td>1000 tips/bag; to dilute antibiotic stocks</td>
</tr>		</tbody>
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the mods method for diagnosis of tuberculosis and multidrug resistant tuberculosis

活動性肺結核の患者（TB）は、患者を硬化し、新たな感染症の予防の両方に不可欠な活動性結核を診断すること、毎年10月15日他の人に感染する。さらに、耐性結核（MDRTBを）多剤の出現は、その薬剤耐性の検出は、薬剤耐性菌の拡散を阻止するために必要であることを意味する。顕微鏡観測薬剤感受性（MODS）アッセイは、TBとMDRTBの高性能の検出のための低コスト、ローテクのツールです。結核菌（MTB）は、微細なMTBの成長は、固形培地上のコロニーの肉眼的外観を待っているよりも早期に液体培地で検出し、可能な）固形培地2に比べて液体培地でより速く成長する1）：MODSのアッセイは、3つの原則に基づいていますその成長は、サブカルチャーは、実行をする必要がない、それは薬イソニアジドとリファンピシンがMDRTBの同時直接検出を可能にするためにMODSのアッセイに組み込むことができる非定型抗酸菌や真菌や細菌汚染3）と区別できるように、MTBの特徴です。間接的な薬剤感受性試験。競合する現在の診断は、喀痰塗抹標本と低感度、固体培地の文化、既存の液体培地培養法と法外に高いコスト、およびMDRTBを検出する間接的な薬剤感受性試験のためにサブカルチャーを行うために必要と診断されるまでに長い遅延によって妨げています。対照的に、非独占的なMODSの方法は、結核とMDRTBための高感度を有し、比較的迅速な培養法であり、MDRTBの同時薬剤感受性試験を提供し、リソースが限られた結核のためのテストのためのすぐ下に$ 3の設定にアクセス可能であり、 MDRTB。

Watch this Scientific Journal Video about The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis at JoVE.com

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

顕微鏡観測薬剤感受性（MODS）アッセイは、結核の高性能検出（TB）と多剤耐性結核（MDRTB）用の低コスト、ローテクのツールです。このビデオは、MODSの液体培地培養法を説明します。

結核および多剤耐性結核の診断のためのMODSの方法

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and ...

the-mods-method-for-diagnosis-tuberculosis-multidrug-resistant

Research

JoVE Journal

Biology

18.9K ビュー.  The Warren Alpert Medical School of Brown University. 顕微鏡観測薬剤感受性（MODS）アッセイは、結核の高性能検出（TB）と多剤耐性結核（MDRTB）用の低コスト、ローテクのツールです。このビデオは、MODSの液体培地培養法を説明します。

ビデオ: The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

の注入。ハマダラカの胚は、マラリア耐性の蚊を生成する

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

生物学

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

表現、界面活性剤可溶化、および膜トランスポーター、MexBの多剤耐性タンパク質の精製

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

腱板断型ボトルの培養システムを使用してげっ歯類全胚培養の方法

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

の野生型と観賞用品種を区別するPCRベースのジェノタイピング法チガヤ</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

脂質Bicelle法を用いた膜タンパク質のハイスループット結晶化

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

イメージングのための簡単​​な方法シロイヌナズナ浸潤イメージング媒体としてPerfluorodecalinを使って葉

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

モザイク大人のライブイメージングのためのトマト/ GFP-FLP / FRT方法<em&gt;ショウジョウバエ</em&gt;視細胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

キナーゼ阻害剤に対して耐性変異のスクリーニングおよび検証のための方法

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

酸化ストレスに赤/近赤外光療法の波長および強度の範囲の効果の評価のための手法<em&gt;インビトロ</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸の濾過単離：簡易迅速DNA抽出方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...