この作品は、K INBRE P20GM103418、カンザス大豆委員会 (KSC/PSU 1663 年)、ACS PRF 56629 UNI7 と SS にすべて電源ユニット高分子化学スタートアップ資金によってサポートされます。ビデオと彼の顕著な仕事のため大学のビデオグラファー、ジェイコブ アンセルミ氏に感謝いたします。我々 もありがとう氏のロジャー ・ Heckert と夫人カタ Heckert 研究のための彼らの寛大なサポート。

1. 合成と多パラメトリック磁気蛍光ナノセンサー (MFnS) の機能化。
<ol><li>超常磁性酸化鉄の合成ナノ粒子 (IONPs) <ol><li>IONP 合成に備えて準備次の 3 ソリューション: ソリューション 1: した FeCl 3 (0.70 g) とした FeCl 2 H 2 O (2 mL)、ソリューション 2: NH 4 ああ (2.0 mL、13.4 M) H 2 O (15 mL)、ソリューション 3: ポリアクリル酸 (0.855 g) H 2 O (5 mL).</li>
		<li>は、ソリューション 1 に 2 M 塩酸 (HCl) の 90 μ l 添加しすぐに 875 rpm でボルテックスしながらソリューション 2 を混ぜます。解決策 3 を追加します。60 分のボルテックスを続ける</li>
		<li>1,620 x g に 20 分間遠心遠心 2,880 x g に 20 分間清</li>
		<li>は、透析による最終的な上澄みを浄化します。透析バッグに上澄みを追加 (MWCO 6 &#8722; 8 K)、リン酸緩衝生理食塩水 (PBS) が含まれているビーカーに配置 (pH = 7.4) とスピン バー。それはすべて 2-3 h. 新鮮なバッファーに置き換えて、12 時間浸してみましょう</li>
	</ol></li> <li>IONPs の表面にターゲット固有の抗体の共役 <ol><li>次の 4 つのソリューションを準備: ソリューション a: 5 mg 1-エチル - 3-(3-dimethylaminopropyl) 250 μ L MES のカルボジイミド塩酸塩 (EDC) [2-(N-morpholino) ethanesulfonic 酸] バッファー (0.1 M、pH = 6.8)、ソリューション b: 3 mg N-(NHS) の MES バッファーの 250 μ L (0.1 M、pH = 6.8)、IgG1、リン酸緩衝生理食塩水 (PBS) (pH 7.4) の 225 μ L で、大腸菌 mAb のソリューション c: 5 μ g、IONP ソリューション d: 4 ml (5.0 m モル) を 1 mL の PBS (pH 7.4)。</li>
		<li>追加 2 (N Morpholino) ethanesulfonic 酸 (MES) バッファー溶液をすぐに追加溶液ソリューション d 15 内の小さな単位で s、混合の各付加の後でソリューションを反転します</li>。
		<li>はすぐに 3 分間にわたって少しずつソリューション d ソリューション B を追加する開始します。各付加の後でソリューションを反転してミックスします</li>。
		<li>混合の各付加の後でソリューションを反転ソリューション C を 5 μ L の単位でソリューションに追加します</li>。
		<li>反応室温で 3 時間して一晩 4 ° C で孵化を続行するを許可します</li>。
		<li>PBS を使用する磁気の列を介して結果 Ab 共役 IONPs を浄化する (pH 7.4、決勝 [Fe] を = = 3.5 mmol) 任意の非抗体を削除します。
		<ol><li>PBS (1 mL) で洗浄磁気の列。磁気プレートに列を添付し、IONP ソリューションを追加します</li>。
			<li>は、PBS (1 mL) の磁気コラムを再度洗浄します。磁気プレートから磁気の列を削除します。PBS を列に追加し、ソリューションを収集します。4 ° C でストア</li></ol></li></ol></li> <li>カプセル化蛍光 1, 1 の &#39; - Dioctadecyl - 3,3,3 &#39; 3、&#39;-Tetramethylindocarbocyanine 過塩素酸塩 (DiI) 色素標識抗体 IONPs を使用してのポリアクリル酸 (PAA) のコーティングに、溶媒拡散法。
	<ol><li>、IONPs の 4 mL に追加滴 2.0 μ L で 1100 rpm 連続混合 DMSO の 100 μ L の DiI 色素 (2 mmol) の</li>。
		<li>[Fe] の最終的な鉄濃度を最終的な MFnS ソリューションの作成、PBS に対して得られた溶液 (12 h MWCO 6-8 KDa) を dialyze = 2 モル</li>。
	</ol></li> <li>MFnS 特性 <ol><li>吸光光度分析用プレート リーダー 595 で蛍光性の放出でカプセル化された DiI 染料を検出する nM</li>。
		<li>動的光散乱法による <ol><li>が平均サイズとは MFnS の表面電荷を決定する zetasizer に MFnS ソリューションのサンプルを配置します 。
			注: ここでは、平均サイズと機能 MFnS の表面電荷ことがわかった 77.09 nm と-22.3 mV、それぞれします</li>。
		</ol></li></ol></li></ol> 2。細菌の培養とストックに調製した溶液 <ol><li>細菌培養 <ol><li>水和物の凍結乾燥ペレット (E. 大腸菌 o157: h7) 栄養流体培養基の 1 mL にし、スープの追加 5 mL を追加します</li>。
		<li>寒天培地と連勝するには、このソリューションの適用 100 μ L を使用して滅菌接種ループ、37 ° C 24 時間で孵化が続く</li>
		<li>後 24 h、寒天板から隔離されたコロニーを選択し、15 mL の培養液を追加します。光学濃度値を監視しながら 4-6 時間、37 ° C で孵化させなさい (吸光度 600 nm).</li>
		<li>0.1 の光学密度値を取得した後、培養を停止し、シリアル希薄を実行します</li>。
	</ol></li> <li>シリアル希薄 <ol><li>8 つの滅菌遠心チューブに栄養流体培養基の追加 900 μ L.</li>
		<li>初の管 (希釈 10 -1) に細菌懸濁液の追加 100 μ L.</li>
		<li>最初から取る 100 μ L チューブ 10 -2 の希釈を取得する、2 番目に追加.残りのチューブ、最終希釈 10 -8 で終わるこのパターンを続ける</li>。
		<li>寒天培地プレートを分離し、37 で 24 時間インキュベート各チューブから転送 100 μ L ° C</li>
		<li>次の日は、各プレート上 (CFUs) の単位を形作っているコロニーをカウントします。〜 100 CFUs のプレートを選択します。さらに実験のための対応する希釈を使用 (100 μ L = 100 ~ CFUs). 
		注: ここにそれは 〜 100 CFUs の結果 10 -6 希釈します</li>。
	</ol></li></ol> 3。迅速な検出の E. 大腸菌 o157: h7 MFnS を使用して <ol><li>スパイク 1 ~ 100 CFU 範囲の結果 10 -6 細菌の在庫の増加額で様々 な PBS ソリューション (1 X、pH 7.4 では、300 μ L)。MFnS の一貫した量を追加 (100 μ L) これらのソリューションにします</li>。
	<li>PBS (1 X、pH 7.4 では、300 μ L) のみが含まれている 1 つのベースライン ソリューションを作成し、MFnS (100 μ L).</li>
	<li>37 ° C で 30 分のためのソリューションをインキュベートし、部屋の温度を冷ますします</li>。
	<li>磁気 relaxometer (0.47 テスラ) と各ソリューションの CFUs 基準緩和時間 (T2) の変更を記録する個々 のソリューションを転送します。
	<ol><li>T2 値の変化を記録、PBS と MFnS のみを含むベースライン ソリューションの T2 値を測定することによって開始します。
		<ol><li>磁気でソリューションの場所 relaxometer。それぞれのソフトウェアを開き、選択、&#34; T2 緩和 &#34; 設定を押し &#34; 測定 &#34;</li></ol></li> <li>様々 なスパイクが追加のソリューションの T2 値を測定基準 T2、のコレクションを次。細菌の濃度. 
		注意: T2 に変更、ベースライン スパイク T2 から減算 T2 に相当します</li>。
	</ol></li> <li>磁気 relaxometer からのサンプルを削除し 2880 x g で 10 分間のチューブを遠心分離</li>
	<li>上清を捨て、100 μ L の PBS (1 X、pH 7.4) の細菌のペレットを再懸濁します</li>。
	<li>96 ウェル プレートと 595 でのレコードの蛍光強度をそれぞれの巻き上がりを追加 80 μ L nM 。
	注: テスト繰り返すことができる結果のセクションで説明するように、湖の水、ミルクなど、異なる溶媒を用いた</li></ol>

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このナノテクノロジーの実証済みのアプリケーションは、まだ FDA によって承認されていません。

このプロトコルは、単に完全に機能の MFnS を生成する設計されている可能な限り。ただし、プロトコルの変更、ユーザーの目的に応じて、役に立つかもしれない多くの重要なポイントがあります。たとえば、異なった抗体の使用は他の多くの病原体の対象になります。さらに、このプロトコルは抗体分子をターゲットとしての使用に限定ではありません。ホスト細胞?...

両方の細菌汚染度の永続的な発生市販食品と水の源はますます急速なと特定の診断プラットフォームの必要性を作成しました。1,2より一般的な細菌汚染食料と水の汚染のための責任の一部はサルモネラ、黄色ブドウ球菌、リステリア菌、腸炎ビブリオ、赤痢菌、細菌、大腸菌属です。3,4多くの場合これらの病原体が細菌汚染の結果熱、コレラ、胃腸炎、下痢などの症状。4しばしば水源の汚染が十分にろ過された水へのアクセスなしでコミュニティに抜本的な不利な影響と食品汚染が病気や製品のリコールの努力の偉大な数につながっています。5,6
細菌汚染に起因する病気の発生を減らすために、水と食品を効率的にスキャン販売または消費する前に方法を開発する努力の数がずっとあります。1,7,8,9,10 12ループ lamp 法 (11,, elisa 法、PCR などの3手法ランプ)13,14 15、16,17,18,19,20,21、 22,23,24は、最近様々 な病原体の検出に使用されています。伝統的な細菌の培養方法と比較して、これらの技術、特異性と時間に関してはるかに多く効率的です。しかし、これらの技術はまだ偽陽性とネガ、複雑な手順、コストと戦います。1,3,25細菌検出のための代替方法として多重磁気蛍光ナノセンサー (MFnS) を提案する、まさにこの理由のためです。
これらナノセンサーでの磁気緩和と迅速かつ適確なデュアル検出プラットフォームを可能にする蛍光のモダリティのペアリング方法一緒に一意に。E. 大腸菌o157: h7 を使用すると、サンプルの汚染物質として、分以内として少し 1 CFU を検出する MFnS の能力を発揮します。病原体特異抗体は特異性を上げるため、磁気や蛍光灯のモダリティの組み合わせにより、検出の両方の低と高の汚染範囲の細菌汚染の定量化。16細菌汚染の場合、ナノセンサー、細菌病原体特異的抗体のターゲットの能力のため周辺群発地震します。磁気ナノセンサーと細菌の間のバインドは、磁気鉄のコアと周辺の水のプロトン間の相互作用を制限します。これは磁気 relaxometer で記録された T2 緩和時間の増加を引き起こします。ソリューションにおける細菌の濃度が上昇すると、T2 値が小さい結果、細菌数の増加など、ナノセンサーを分散します。逆に、病原体を直接バインド ナノセンサーの数が増えたため、細菌の濃度に比例した蛍光性の放出が増えます。サンプルの遠心分離と分離細菌のペレットのみ任意の浮遊ナノセンサーを取り外して直接関連付けの数と蛍光性の放出、細菌に直接接続されているナノ粒子を節約します。ソリューションに存在する細菌。このメカニズムの概略は、図 1で表されます。
この MFnS プラットフォームは、ポイント ・ オブ ・ ケア スクリーニング低コスト ・ ポータブルの特性で、心で設計されています。MFnS は常温で安定しているし、細菌汚染の正確な検出のための非常に低濃度で必要なだけです。さらに、合成後に、、MFnS の使用は簡単、分野で訓練を受けた専門家の使用を必要としません。最後に、この診断プラットフォームにより高度なカスタマイズをターゲットに、さまざまな設定のすべての種類の病原体を検出する 1 つのプラットフォームを使用可能性があります、この手段を提供します。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ferrous Chloride Tetrahydrate</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">I90-500</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ferric Chloride Hexahydrate</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td>I88-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ammonium Hydroxide</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">A669S-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Hydrochloric Acid</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">A144S-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Polyacryllic Acid</td>
			<td style="width:156px;">Sigma-Aldrich</td>
			<td style="width:197px;">323667-100G</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">EDC</td>
			<td style="width:156px;">Thermofisher Scientific</td>
			<td style="width:197px;">22980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">NHS</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td>AC157270250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-E. coli O111 antibody&#160;</td>
			<td style="width:156px;">sera care</td>
			<td>5310-0352</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-E. coli O157:H7 antibody [P3C6]&#160;</td>
			<td style="width:156px;">Abcam</td>
			<td style="width:197px;">ab75244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">DiI Stain</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">D282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Nutrient Broth</td>
			<td style="width:156px;">Difco</td>
			<td style="width:197px;">233000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freeze-dried E. coli O157:H7 pellet</td>
			<td style="width:156px;">ATCC</td>
			<td style="width:197px;">700728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Magnetic Relaxomteter&#160;</td>
			<td style="width:156px;">Bruker</td>
			<td style="width:197px;">mq20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Zetasizer</td>
			<td style="width:156px;">Malvern</td>
			<td style="width:197px;">NANO-ZS90</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Plate Reader&#160;</td>
			<td style="width:156px;">Tecan</td>
			<td style="width:197px;">Infinite M200 PRO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Magnetic Column&#160;</td>
			<td style="width:156px;">QuadroMACS</td>
			<td style="width:197px;">130-090-976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Centrifuge</td>
			<td style="width:156px;">Eppendorf</td>
			<td style="width:197px;">5804 Series</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Centrifuge (accuSpin Micro 17)</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">13-100-676</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Floor Model Shaking Incubator</td>
			<td style="width:156px;">SHEL LAB</td>
			<td style="width:197px;">SSI5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Analytical Balance</td>
			<td style="width:156px;">Metler Toledo</td>
			<td style="width:197px;">ME104E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Digital Vortex Mixer</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">02-215-370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Open-Air Rocking Shaker</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">02-217-765</td>
			<td></td>
		</tr>
	</tbody>
</table>

foodborne pathogen screening using magneto-fluorescent nanosensor: rapid detection of e. coli o157:h7

腸管出血性大腸菌o157: h7 は、水性の両方にリンクされている媒介する病気と食料と水上映方法にも関わらず脅威は現在使用されるまま。従来の細菌検出方法、ポリメラーゼ連鎖反応 (PCR)、酵素抗体法 (ELISA) 特に病原性の汚染物質を検出できるよう彼らが豊富なサンプル準備と待機時間が長く必要です。さらに、これらのプラクティスは洗練された実験室の器械と設定、要求し、訓練を受けた専門家によって実行される必要があります。ここで、ナノ粒子ベースのプラットフォームの磁気および蛍光パラメーターの一意の組み合わせを備えて簡単診断技術のためのプロトコルを提案します。提案多重磁気蛍光ナノセンサー (MFnS) は、大腸菌o157: h7 汚染としてはほとんど 1 コロニー形成単位未満 1 時間以内のソリューションに存在を検出できます。牛乳など複雑なメディアで高機能を維持する MFnS の能力をさらに、湖の水を確認します。その他特異性アッセイも同じような細菌種の存在下でも、特定のターゲット細菌のみを検出する MFnS の能力を発揮する使用されました。検出の両方の初期と後期段階の汚染検出でその高性能を出展、濃度の広い範囲の病原体汚染の定量化により、磁気や蛍光灯のモダリティのペアリングします。有効性、手頃な価格、および、MFnS の移植性から作ることがポイント ・ オブ ・ ケア スクリーニング細菌汚染のための理想的な候補の設定の広い範囲で市販加工食品に水生の貯水池。

MFnS の作用機序は、図 1で表されます。細菌汚染物質の表面周囲の MFnS のクラスタ リングは、MFnS の magnetic cores と周囲の水素原子核の相互作用を妨げます。このクラスタ リング、磁気緩和の結果として値が増加します。細菌汚染の濃度が増えると、クラスタ リングが縮小し、T2 値の変動が減少します。したがって、蛍光のモダリティの追加が重要です。細菌の濃度が増...

Watch this Scientific Journal Video about Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7 at JoVE.com

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

このプロトコルの全体的な目標はポータブル、コスト効果の高い、機能的なナノセンサーを合成する、特にターゲットに磁気緩和と蛍光発光モダリティの組み合わせにより病原性細菌の迅速検出。

E. 大腸菌o157: h7 の食品由来病原体のスクリーニングを使用して磁気蛍光ナノセンサー: 迅速検出

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

foodborne-pathogen-screening-using-magneto-fluorescent-nanosensor

Research

JoVE Journal

Immunology and Infection

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

7.6K ビュー.  Pittsburg State University. このプロトコルの全体的な目標はポータブル、コスト効果の高い、機能的なナノセンサーを合成する、特にターゲットに磁気緩和と蛍光発光モダリティの組み合わせにより病原性細菌の迅速検出。

ビデオ: Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

粘着テープベースのサンプリングと蛍光の組み合わせその場で</emの迅速検出のための>ハイブリダイゼーションサルモネラ生鮮食品について

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

免疫・感染学

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

細胞運命を以下に<em&gt; E大腸菌の</emラムダファージによる感染後&gt;

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

真菌病原体の食作用の生細胞ビデオ顕微鏡

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV逆転写酵素及びインテグラーゼ阻害剤の迅速なスクリーニング

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

の種特異的検出のためのLAMP法（LAMP）アッセイ<em&gt;アイメリア</em&gt;鶏に感染する

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

インフルエンザウイルスAおよびBの急速な分子の検出と分化

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

密なトランスポゾン挿入ライブラリを使用して細菌活用腸内細菌系統など、大腸菌や赤痢菌の作成

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

迅速・安全・簡易マニュアル ベッドサイド核酸抽出の全血試料中のウイルス検出

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

サルモネラ属菌のスクリーニングのための高スループット プラットフォーム/赤痢菌属

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.