この研究は、韓国国立研究財団からの助成金（2013R1A1A3005669）と韓国の医療技術のR＆Dプロジェクト、保健福祉省の補助金（HI13C0016）によってサポートされていました。

1.プローブの準備<ol><li>プローブのPCR増幅<ol><li> Pのジンジバリス特異的プローブは、Pの343 bpのDNA断片を増幅しますPのゲノムDNAを用いたPCRによるジンジバリスの16SrRNA ジンジバリスと以下のプライマー：5&#39;-TGC AAC TTG CCT TAC AGA GG-3 &#39;および5&#39;-ACT CGT ATC GCC CGT TAT TC-3&#39; 10。 72℃で10,11で5分間の延長が続く1分20秒、30秒、30秒、95℃で35サイクル、60℃、72℃以下のサイクル条件で増幅を行います。 </li><li> 70-bpのDNA断片（5&#39;-CAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGを使用して、真正細菌のプローブを増幅 TCCCGCAACGAGCGCAACCC-3 &#39;オリゴヌクレオチドおよび以下のプライマーとして合成さ）：5&#39;-CAG GTR CTG CMT GGY-3&#39; &#39;11および5&#39;-AGG GTT GCG CTC GTT-3。 40サイクル：次のサイクル条件で増幅を行います30秒30秒9、4℃、60℃、1分間72℃11,12で5分間の延長に続く20秒間の72℃で。 </li><li> 2時間-20℃でインキュベートし、3M酢酸ナトリウム（pH5.2）と（：1：2の比率10で）100％エタノールを添加することにより、PCR産物（≥500μl）を沈殿させます。 </li><li>遠心分離機15分間14,000×gで混合物を、100μlのTE緩衝液にペレットを再懸濁し、260 nmでの光学濃度によりDNA濃度を測定します。 </li></ol></li><li> DIG DNA標識および検出キットを用いてDIGは、標識<ol><li> 15μlの最終容量まで水でプローブの3μgのを混ぜます。 </li><li> 10分間95℃でDNAを変性し、すばやく氷上で冷やします。 </li><li>変性DNAの15μlに10倍のヘキサヌクレオチドミックス2μlの、のdNTP標識ミックス2μlの、およびクレノウ酵素の1μlを添加します。 </li><li>混合し、48時間に24時間、37℃でインキュベートし、65℃で加熱することによって反応を停止させます。 </li></ol></li><li> DIG DNA標識および検出キットを用いてDIG標識プローブの感度を確認し<ol><li>手動で室温で5分間、キットに提供ポジティブコントロールプローブの連続希釈液を1μl、ナイロン膜上のDIG標識プローブ1ngの、乾燥をロードします。 </li><li>簡潔には2×SSC緩衝液（0.3MのNaCl、30mMのクエン酸ナトリウム、pH7.0）中の膜を浸漬し、DNAを固定化するために1時間80℃で膜をインキュベートします。 </li><li>提供ブロッキング溶液6mlに：簡単に言うとマレイン酸バッファー（MAB、0.1 Mマレイン酸、0.15 MのNaCl、pH7.5）で、膜を浸して、抗DIGアルカリホスファターゼ（AP）でインキュベートする抗体希釈（5000 1）抱合30分間、室温でキット中。 </li><li> MABT（MABを1％Tween 20を含む）を有する膜を洗浄し、上に2ミリリットルの検出緩衝液（0.1 Mトリス塩酸、0.1 MのNaCl、0.05 MのMgCl 2、pHは9.5）と混合し、NBT / BCIP溶液40μlを適用1分間の膜。標識プローブの感度がある場合1.1.3から1.3.4へのステップを繰り返し、満足しません。 </li><li>標識プローブ（OD260）のDNA濃度を測定し、μlのngの100の濃度に調整-1。 </li></ol></li><li> DIG標識プローブの特異性を確認してください<ol><li> 10分間95℃で、特異的プローブによって標的細菌を含む様々な細菌種からのゲノムDNA試料（25ngの3μL-1各サンプルあたり）を変性させ、すぐに氷上で冷やします。 </li><li>手動で室温で20分間、変性ナイロン膜上のDNAと乾燥をロードします。 </li><li>簡単に言うと2×SSC緩衝液中で膜を浸して、DNAを固定化するために80℃で2時間、膜をインキュベートします。 </li><li> 1時間室温でブロッキング溶液で膜をブロックします。 </li><li> 、ハイブリダイゼーション緩衝液中で-1μlのngの1でプローブを希釈し希釈したプローブを変性させ、膜上に適用されます。 </li><li> 1時間45℃で膜をインキュベートします。 </li><li>メンブレンを洗浄32×SSC緩衝液で倍。 </li><li>簡単に言うとMABに膜を浸します。 </li><li>室温で1時間ブロッキング溶液6mlに：（2000 1）希釈した抗DIG APコンジュゲート抗体とインキュベートします。 </li><li> MABTでメンブレンを洗浄し、2ミリリットルの検出緩衝液を混合し、NBT / BCIP溶液40μlを適用します。 </li><li>プローブが予想特異性を達成していない場合の特異性が観察されるまで、ハイブリダイゼーション温度を上昇させます。 </li></ol></li></ol> in situハイブリダイゼーション 2 注：検体と試薬の乾燥を避けるために、湿ったペーパータオルを敷いた加湿チャンバー内で全てのインキュベーションを行います。 <ol><li>組織切片の脱パラフィン除去し、再水和<ol><li>パラフィン包埋ブロックから厚さ4μmの組織切片を準備します。ホルマリン、パラホルムアルデヒド、及び亜鉛系固定液は、このプロトコルに対応しています。 </li><li> DEPC処理水を用いて、全ての試薬を準備します。 </li><li> 30分間60℃で乾燥オーブン中で加熱し、スライドを室温で30分間スライドをインキュベートします。 </li><li> 5分間3回、100％キシレンでスライドを浸します。新鮮なキシレンを毎回使用しています。 注：化学ドラフト内でこの脱脂の手順を実行します。 </li><li>新鮮なエタノールを毎回使用して、二回5分間、100％エタノールでスライドを浸し、シリアル、90％、80％、5分間、それぞれ70％エタノール溶液中の試料を再水和。 </li><li> 1分間のDEPC処理水にスライドを浸し、6分間DEPC処理PBS（pH7.4）中でスライドを洗浄します。 </li></ol></li><li>組織切片の前処理<ol><li> 20分間0.1 N塩酸でスライドを浸し、30秒間DEPC処理水でスライドを洗浄します。 </li><li>ワックスペンを使用して、試験片の周囲の疎水性の障壁を描画します。 注：疎水性の障壁の大きさは、標本の大きさに依存します。したがって、次の手順で使用される試薬の体積は試料のサイズに応じて異なるが、HYを充填する必要がありますdrophobicバリア（小標本と大きな標本のために400μlの50μL）。 </li><li> 30分間37℃で乾燥オーブン中でプロテイナーゼKの50〜400μlの（1から10μgのml -1のDEPC処理したPBS中）を用いて検体を処理し、1分間、DEPC処理した1×PBSでスライドを洗浄します。 </li><li> 1分間のDEPC処理PBSでスライドを洗浄し、その後、10分間、DEPC処理したPBS中の4％パラホルムアルデヒド中にスライドを浸します。 </li><li> 20分間0.5％の無水酢酸を含む0.1Mトリエタノールアミン - 塩酸（pH8.0）でのスライドを浸し、30秒間DEPC処理水でスライドを洗浄します。 </li></ol></li><li>プローブと洗浄とのハイブリダイゼーション<ol><li> 20分間、2×SSC緩衝液でスライドを浸し。 </li><li> 1 ngのμL-1のハイブリダイゼーション緩衝液（4×SSC、50％[体積/体積]ホルムアミド、1×デンハルト溶液、10％[重量/容量]デキストラン硫酸、0.1％[重量/容量]ドデシル硫酸ナトリウムでプローブを希釈0.4ミリグラムml -1のサケ精子DNA）。陰性対照のために、で標識したプローブをミックス非標識プローブの10倍過剰量。 </li><li>希釈されたプローブを変性させ、組織切片上に50〜400μLを適用します。スライドにカバースリップを置き、マニキュアで密封します。 </li><li> 45℃または最適な温度での加湿チャンバーのO / Nで10分間、90℃でのPCR装置内の熱スライドとインキュベートスライドはステップ1.4.11で決定されました。 </li><li> 、4℃で30分間スライドを冷却カバースリップを削除し、以下のようなシリアルSSC緩衝液でスライドを浸す：10分間の4×SSC、10のために20分間予熱した（45℃）2×SSC、2×SSC分、および10分間の0.2X SSC。 </li></ol></li><li>抗DIG AP結合抗体で検出<ol><li> 5分間MABTでスライドを洗浄します。 </li><li> 20分間、1％Tween 20でブロッキング溶液の50〜400μlのブロック。 </li><li>溶液（1：1,000）をブロックに希釈した抗DIG-AP抗体の50〜400μl添加しanld 37℃で90分間インキュベートします。 </li><li> 10分間MABTでスライドを洗浄します。 </li> &lt;Liが&gt; 5分間、1％Tween 20を含む検出緩衝液中でスライドを浸し。 </li><li> μ1mMのレバミゾールの内因性アルカリホスファターゼを不活性化するために5分間（1％Tween 20を含む検出緩衝液中で）50〜400で処理します。 </li><li>各試料にと開発された信号が良好になるまで2〜3時間、室温で加湿チャンバー内でスライドをインキュベート：（100 1で検出緩衝液で希釈）予混合NBT / BCIP溶液を50〜400μLを配布します。 </li><li>可視化を停止し、10分間37℃で0.05％カウンター染色として[重量/容量]メチルグリーンの50〜400μLを適用し、脱イオン水でスライドをすすぎます。 </li><li> 、脱イオン水でスライドを洗浄し、100％エタノール中で脱水し、キシレン中に浸します。 </li><li>各スライド上に取り付け、溶液の80μLを適用し、カバースリップでカバーしています。 </li></ol></li></ol>

<ol>
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著者らは、開示することは何もありません。

ここで、DIG標識DNAプローブを用いてパラフィン包埋組織内の細菌をローカライズするためのプロトコルが記載されています。プローブは、細菌の16S rRNA遺伝子のDNAまたはRNA分子を標的とし、16S rRNAの標的プローブは、いずれかの種特異的または普遍として設計することができます。 P.の特異的ハイブリダイゼーションP.ジンジバリスに対する特異的プローブジンジバリスで?...

細菌は、歯周炎、歯髄炎、歯冠周囲炎、蜂巣炎、骨髄炎などの様々な口腔疾患の病因における役割を果たしています。疾患の病因における細菌の役割を理解し、治療の効果を監視するために、組織内の細菌の局在化は重要です。歯周炎患者からの歯肉組織内の細菌の存在は、蛍光-を用いたin situハイブリダイゼーション （FISH）8 の細菌に特異的な抗体3-7、および蛍光を用いて、電子顕微鏡1,2を含む様々な方法を用いて、免疫組織化学および免疫蛍光が示されています16S rRNAのをターゲットに標識されたオリゴヌクレオチドプローブ。それにもかかわらず、これらの方法は広く、技術的制限または困難に使用されていません。抗体と比較して、16SのrRNAを標的とするプローブを生成し、種特異性を達成することは容易です。 FISHはBACTの可視化のための優れたツールであることが証明されましたこのようなプラークバイオフィルムとしての自然環境でERIA。しかし、組織サンプルのFISHの適用は、様々な組織成分の自己蛍光に限定されます。彼らは9出血含むとき、例えば、赤血球の強い自己蛍光は、しばしば、炎症組織への蛍光技術の適用を妨げます。 炎症を起こした歯肉組織内の細菌を局在化するためには、従って、ジゴキシゲニン（DIG）を用いたin situハイブリダイゼーション法での DNAプローブの開発に成功10,11に適用された標識。 Pを使用してパラフィン包埋組織内の細菌の局在化のためここでは詳細なプロトコールジンジバリス特異的およびユニバーサル真正細菌プローブが記載されています。同様の結果は、他の研究室で再現することができるように、これは、特に方法の標準化に焦点を当てています。このプロトコルは、その組織学的コンテキストとRESU内細菌の局在化を可能にしますLTSは非常に再現性があります。説明されたプロトコルのいずれかの様々な組織における種特異的または普遍方法で細菌をローカライズするために使用することができます。ユニバーサルプローブは、多菌性疾患中の細菌を検出すると、特定の細菌の役割は知られていない疾患における細菌の潜在的な役割を研究するために特に有用です。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Acetic anhydride</td>
			<td>Sigma</td>
			<td>6404</td>
			<td></td>
		</tr>
		<tr>
			<td>50% Dextran sulfate solution</td>
			<td>Millipore</td>
			<td>S4030</td>
			<td></td>
		</tr>
		<tr>
			<td>50X Denhardt&#8217;s solution</td>
			<td>Sigma</td>
			<td>D2532</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC</td>
			<td>Sigma</td>
			<td>P159220</td>
			<td></td>
		</tr>
		<tr>
			<td>DIG DNA labeling and detection kit</td>
			<td>Roche</td>
			<td>11 093 657 910</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma</td>
			<td>F9037</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmEdge&#8482; Pen</td>
			<td>Dako</td>
			<td>H-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>Vector</td>
			<td>SP-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma</td>
			<td>246964</td>
			<td></td>
		</tr>
		<tr>
			<td>Maleic acid</td>
			<td>Sigma</td>
			<td>M0375</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl green</td>
			<td>Sigma</td>
			<td>M6776</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P1648</td>
			<td></td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon sperm DNA solution</td>
			<td>Invitrogen</td>
			<td>#15632-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>S9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Duksan</td>
			<td>D1420</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Amresco</td>
			<td>227</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethanolamine-HCl</td>
			<td>Sigma</td>
			<td>90279</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Research organics</td>
			<td>3098T</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ detection of bacteria within paraffin-embedded tissues using a digoxin-labeled dna probe targeting 16s rrna

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

図1に示すように、それらの感受性を決定するためのキットで提供ポジティブコントロールプローブと比較してDIG標識プローブのドットブロッティング。 343 bpのP.ジンジバリス特異的プローブは、70塩基対真正細菌プローブよりも25倍より敏感である。Pの検出のための慢性歯周炎の患者から得られた歯肉組織のin situハイブリダイゼーション 図2図</...

Watch this Scientific Journal Video about In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA at JoVE.com

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

16S rRNAのをターゲットジゴキシン標識DNAプローブを用いたパラフィン包埋組織内細菌のその場検出で

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

in-situ-detection-bacteria-within-paraffin-embedded-tissues-using

Research

JoVE Journal

Immunology and Infection

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

10.9K ビュー.  School of Dentistry and Dental Research Institute, Seoul National University. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

ビデオ: In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

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

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

インフルエンザのためのDNAマイクロアレイ上ampliPHOX比色検出

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

オリゴヌクレオチド結合蛍光マイクロスフェアを使用して複雑な臨床及び環境試料中の細菌の多重検出

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

蛍光性DNAzymesを使用した細菌の検出

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms&#160;and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5&#39; or 3&#39; ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections.

蛍光<em&gt;その場での</emウイルスと植物及び昆虫組織における共生細菌の局在化のための&gt;ハイブリダイゼーション（FISH）

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

MALDI-TOF質量分析法を使用した血液培養ブロスからのグラム陰性菌の迅速な同定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

主要組織適合遺伝子複合体クラスII Dextramersを用いて脳および心臓において自己反応性CD4 T細胞のその場検出に

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

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 ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

16S rRNAの遺伝子配列決定およびMALDI-TOF MSによるレア細菌性病原体の同定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

A Method for Targeted 16S Sequencing of Human Milk Samples

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as with all these technologies, reproducible results depend on a laboratory workflow that incorporates appropriate precautions and controls. This is particularly important with low-biomass samples where contaminating bacterial DNA can generate misleading results. This article details a semi-automated workflow to identify microbes from human breast milk samples using targeted sequencing of the 16S ribosomal RNA (rRNA) V4 region on a low- to mid-throughput scale. The protocol describes sample preparation from whole milk including: sample lysis, nucleic acid extraction, amplification of the V4 region of the 16S rRNA gene, and library preparation with quality control measures. Importantly, the protocol and discussion consider issues that are salient to the preparation and analysis of low-biomass samples including appropriate positive and negative controls, PCR inhibitor removal, sample contamination by environmental, reagent, or experimental sources, and experimental best practices designed to ensure reproducibility. While the protocol as described is specific to human milk samples, it is adaptable to numerous low- and high-biomass sample types, including samples collected on swabs, frozen neat, or stabilized in a preservation buffer.

A semi-automated workflow is presented for targeted sequencing of 16S rRNA from human milk and other low-biomass sample types.

人間のミルクのサンプルのターゲット 16S シーケンス法

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as ...