The work carried out in this study was supported in part by the Royal Veterinary College through the student research projects fund, as well as the Biotechnology and Biological Sciences Research Council and the Department for International Development (grant number BB/H009337/2). This manuscript has been assigned the reference PPB_00795 by the RVC.

1.テンプレートの調製注：ニワトリに感染7 アイメリア種のうちの1つに由来するDNAを含有することが疑われる任意のゲノムDNA鋳型は、LAMPベースのアイメリア種同定のための鋳型として使用することができる。ここで説明したように、フィールド診断解析のための腸組織サンプルは、ルーチン、死後の間に収集されるべきである。 <ol><li>試験対象の腸のセクション（またはセクション）を選択します。サンプルサイトの選択へのガイド、サンプリング部位の分布と場所の種特異的な範囲のための18に存在し、 図1である可能性が最も高いアイメリア種については、表1を参照してください。 NOTE： アイメリアは、特にホストサイト特異的であるので、これは極めて重要である。鶏に感染するそれぞれの種は、それが19を対象と腸の地域によって定義されます。決定は、1つまたはカ月の前の農場の経験、関心によって影響され得る特定のアイメリア種または他の診断指標6再。 </li><li>物品5cm以上のEimeria滅菌ハサミ又は外科用メスを使用してテストするために選択された腸の部分（単数または複数）のより長い長さ。必要に応じて、そのようなRNAlaterで18または95％エタノール中で例えばとして固定液中でその後の分析のためのサンプルを格納。 注：サンプルは使用前に無菌トリス - エチレンジアミン四酢酸（TE）バッファーで十分に洗浄する必要があり、エタノールに保存します。 </li><li>縦方向に開いた試料を切断、ほとんどの腸の内容（存在する場合）を削除し、滅菌ガラス顕微鏡スライドまたはエタノール/火炎滅菌した鋏ブレードのエッジのいずれかを使用して、粘膜層を無料から細胞をこすり取る。必要に応じてプールしたサンプルのための単一のチューブ内で種特異的腸内のすべてのサイト4からの細胞を含む。 </li><li> 1を含む100μlの滅菌TE緩衝液を含む無菌の1.5ミリリットルのスクリュートップマイクロ遠心チューブに掻き材料を入れる0％（w / v）のキレックス100樹脂。 </li><li> 1分間激しく、各サンプルを振る。スクリュートップがしっかりと閉じていることを確認した後、10分間沸騰水浴中でインキュベートする。 </li><li>煮沸後、各サンプルは、1~2分間、周囲温度で冷却する。 </li><li> 1分間遠心操作し、最高速度（ 例えば、〜を10,000×g）でのマイクロ遠心を使用して、各サンプル。 </li><li>各LAMPアッセイにおけるテンプレートが着手されるように得られた上清2μlのを収集します。必要に応じて、マルチサイト試験を提供するために、単一のチューブで複数の腸の部位をプールする。 </li></ol> 2. アイメリア LAMPプライマーの準備（プリ検定） <ol><li> 100アッセイ用の適切なアイメリア LAMPプライマーの株式を準備します。 <ol><li> （製造業者で指定された）、100μMの濃度に分子グレードの水を添加することにより、各凍結乾燥アイメリア LAMPプライマーを再構成する。指定しない場合、分子グレードの水必要な使うの体積を計算グラム各プライマーの物理的および分子量。 </li><li>各アイメリア種ごとに個別の0.5ミリリットルフリップトップのマイクロ遠心チューブにピペットで60μlの分子グレードの水をアッセイする。 </li><li>追加のプライマーFIP、BIP、F3、B3、7種特異的プライマーミックスのシリーズを作成し、表2に示したボリュームを用いて水に目標アイメリア種に特異的なLF及びLB、。 </li><li>簡単に言えば渦はその後、プライマー溶液を混ぜてマイクロフュージをパルスし、必要になるまで凍結。 </li></ol></li><li>アッセイすべき各アイメリア種のためのLAMP反応マスターミックスを準備します。サンプル数で、表3に示すボリュームを乗算し、陽性対照、陰性対照及びピペットスペア3つの追加。 0.5または1.5ミリリットル中にピペットフリップトップマイクロ遠心チューブ。 </li></ol> 3. アイメリア LAMPアッセイ<ol><li>ピペット23μlのアイメリア種特異的のBst DNAポリメラーゼ/ LAMPマスターミックス0に。5ミリリットルマイクロ遠心チューブ。 </li><li> 25μlの最終反応容量を作る、（セクション1で調製した）2μlのDNAテンプレートを追加します。 </li><li>一つの反応（陽性対照）に2μlのアイメリア種特異的ゲノムDNAを追加します。反応（ネガティブコントロール）に2μlの分子グレードの水を追加します。 NOTE：種特異的ゲノムDNAが使用できない場合、以前の正のLAMPまたは標準的なPCR産物を代わりに使用してもよい。 </li><li> 30分間62°Cのの水浴またはヒートブロックでインキュベートする。反応はすぐに読み取られるつもりはない場合は、必要に応じて、10分間、80°Cのに加熱することにより、Bst DNAポリメラーゼを非活性化する。 </li></ol> 4. LAMPアッセイ読み取りアウト<ol><li>インキュベーションの終わりには、室内光下での目による各反応の色を評価する。陰性の結果は、陽性の結果が空20青く見える色で紫色ピンク色に見える。 </li><li>任意に、laboratにおけるLAMPアッセイ結果を確認1×トリス/ホウ酸塩/ EDTA（TBE）緩衝液中の2％アガロースゲルを用いたアガロースゲル電気泳動のための1μlのDNAのゲルローディング緩衝液と5μlのLAMP反応生成物を混合することによりモリ（51μl当たり核酸染色を用いて事前に染色アガロース50ml）で。フラグメントサイズの計算を可能にするために、ゲルの1レーンが1KB分子サイズのDNAラダーの5μlを添加する。 </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

The Eimeria species-specific LAMP assays described in this paper offer a new diagnostic tool kit in support of effective control of coccidia and the disease coccidiosis. The outcomes of eimerian infection can include severe economic loss as well as seriously compromised bird welfare and increased susceptibility to colonisation by zoonotic pathogens21. Opportunities to monitor flocks for the occurrence of some, or all Eimeria species can provide early warning of a breakdown in anticoccidial control efficacy. Key advantages of LAMP include robust target specificity, resulting from the requirement for six different DNA sequence targets, as well as high sensitivity, boosted by the inclusion of loop primers13, although the qualitative, not quantitative nature of LAMP may be considered a limitation. It is not currently possible to discriminate low level parasite escape from routine chemoprophylaxis or live vaccine replication from unchecked eimerian replication. Nonetheless, the technical ease of the protocol and definitive readout offers considerable improvement over the existing specialist and frequently subjective pathology- and morphology-led approaches6,7. Each assay may be completed at a cost of ~&#163;0.75 per sample, independent of labour and equipment set up expenses. Thus, LAMP assays are also more cost effective than other molecular diagnostics such as PCR, since they require an isothermal incubation with no need for specialist equipment. 
For many years access to Eimeria genomic DNA as template has limited the development and application of molecular field diagnostics. The oocyst is the most readily accessible phase of the eimerian lifecycle, but routine DNA extraction requires laboratory facilities22. Other, more labile intestinal lifecycle stages require purification prior to DNA preparation to prevent PCR inhibition and a consequent loss of sensitivity23,24. The ability to extract eimerian DNA of a quality suitable for LAMP using equipment no more specialised than a microcentrifuge and a water bath, supplemented by inhibitor adsorption using chelex resin, now promotes the wider use of molecular biology in eimerian diagnostics. Intriguingly, the reported detection of quantitative PCR-measurable Eimeria DNA in intestinal tissue 20 days after the initiation of parasite infection, 11 days after the last detectable oocyst output, raises the suggestion that LAMP may be used to detect resolved parasite exposure as well as ongoing infection, even after any visible lesions may have been resolved25. 
The relatively low cost and low technical requirements of LAMP Eimeria diagnostics can promote their application in the developing world where other more established approaches may not be available or appropriate. For this to be applicable each assay must be capable of detecting all strains which may be circulating within each region. While understanding of the genetic diversity prevailing among Eimeria species is limited26, the use of target sequences previously validated for use in quantitative PCR with strains from Africa, Asia, Europe and South America provides some evidence of conservation, supporting the utility of these LAMP assays around the world10.

グローバル鶏の生産は、発展途上国は、先進国で目撃4倍近く拡大ホスティングで最後の50年間で10倍に増加している<a href="http://www.faostat.org">（www.faostat.orgを）</a> 。世界の食料安全保障への鶏肉生産の関連性もそう成長したようにニワトリで重大な病気を引き起こす可能性が病原体のプロフィールを有する。一典型的な例アイメリア種 、腸疾患のコクシジウム症1を引き起こす可能性がありますユビキタス寄生原虫である。どこ鶏は、一つ以上のアイメリア種が共通2-4である可能性が高いで飼育されている。開発された世界ではアイメリアは、主に抵抗5の影響を最小限に抑えるために、シャトルや回転プログラムを採用し、予防的化学療法によって制御されます。生ワクチンはまた、鳥の値がコストを正当化するのに十分なシステムで使用される（ 例えば、株式、層、およびいくつかのブロイラー5繁殖 ）。 RESUとしてサブ臨床感染は5一般的ですが、これらの措置のLT臨床コクシジウム症は、多くの場合、十分に制御される。発展途上国のワクチン接種ではまれであり、薬物のアプリケーションが頻繁にあまり知らさ。その結果、サブ臨床および臨床のコクシジウム症は、より一般的であり、重大な経済的影響3を発揮する。 最も広く使用されているスコアリングシステムのさえ著者はいくつかの種のために「それはそのような手順は、任意のが、適度に重篤な感染で試みすべきかどうかは疑わしいようだ」とコメントしたがeimerian感染の診断は、伝統的に、死後に得点病変に依存してきた6。形態をオーバーラップすると、すべてが、専門家6,7を混乱することができますが、補足的証拠は、糞便やごみのサンプルで耐環境性オーシストのライフサイクルステージの顕微鏡検出を通じて収集することができます。ポリメラーゼ連鎖反応（PCR）、ランダムamplificatioを用いた分子の代替多型DNA PCR（RAPD-PCR）および定量PCR技術のnは最大20年8月10日のために利用されているが、現在までに、彼らは一般的になってすることができなかった。相対費用と専門実験装置や処理のための要件は、古いpathology-と顕微鏡ベースの、多くの場合、主観的かつ技術的に要求の厳しい自然が10,11に近づくにもかかわらず、それらの取り込みを制限している。このような制限は、貧困にコクシジウム症の影響は12比例して大きくすることができるような東南アジアなど世界の貧困地域の多くでは誇張することができます。これに応答して、 アイメリア種特異的診断アッセイ新しい簡単で敏感が、コスト効果の明確な要求がある。 LAMP法（LAMP）は、大量のDNAを増幅することができるDNAポリメラーゼドリブン技術を準備することは容易である。最も重要なのは、LAMP BST は DNA polymeraを使用していますそれに代えて、熱サイクル13,14を必要とすることなく、単一の一定温度でDNA増幅を促 ​​進する一般的PCRで使用したTaq DNAポリメラーゼ。 LAMPは、最も初歩的な実験室でするか、フィールドにアプリケーションに適用可能であり得る。多くのPCR阻害物質、高い感度と特異性に対する抵抗性に特徴、LAMPアッセイは、伝染性ファブリキウス嚢病ウイルス、 ウェルシュ菌及びクリプトスポリジウム 15-17を含む病原体の広い範囲のために開発されている。応答では、鶏に感染する7 アイメリア種のそれぞれに固有のLAMPアッセイのパネルが18を開発された新たな費用対効果のアイメリア種固有の診断のために要求する。新しいアッセイのためのアプリケーションは、そのような貧しい経済のPEとのEimeriaマキシマやアイメリア·ネカトリックスとして種の関連付けを与えられた特定の値の監視寄生虫の発生を含み、rformance 3,4。他のアプリケーションは、ファームの抗コクシジウム戦略、サブ臨床感染または臨床疾患の診断とファームにアイメリアによるリスクの評価の有効性を評価する含まれています。

<table border="0" cellpadding="0" cellspacing="0" width="889">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Name</td>
			<td style="width:161px;">Company</td>
			<td style="width:139px;">Catalogue number</td>
			<td style="width:295px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">RNAlater</td>
			<td style="width:161px;">Ambion</td>
			<td style="width:139px;">AM7024</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Ethanol</td>
			<td style="width:161px;">VWR Chemicals</td>
			<td style="width:139px;">20821.321</td>
			<td style="width:295px;">Caution, highly flammable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">100 x Tris-EDTA (TE) buffer concentrate</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">T9285</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Chelex 100 resin</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:139px;">142-1253</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Molecular grade water</td>
			<td style="width:161px;">Invitrogen</td>
			<td style="width:139px;">10977035</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. acervulina F3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CCTAACATTTCGCTTCACGGAC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. acervulina B3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*ATGAGCAAGTGGAACACCTTG</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. acervulina FIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*AGAGCACAGTGGCAGTGC-AGCAGACAGCATGGCTTACCT</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. acervulina BIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GAAGACCCTCTGAAGAACGGA-CCTTCTCACCGCTTACCGG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. acervulina LB</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*TAAGGTTACACCCGTGGAGG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. acervulina LF</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GCCATGCACAAAGCGACTT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. brunetti F3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GGCCATCAAGTTCCATGAGC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. brunetti B3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*TCAACCTCCTGAGTGTGGTT</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. brunetti FIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GAAAATGCCTTCGTAGCTGCT-GCTGGGTACGGAGCGTCTT</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. brunetti BIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*TACTTCCTAGGATCCATCCTCGC-AGTTTCGCTGCCGCCTC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. brunetti LB</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GAAACGCTCGAACATGGC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. brunetti LF</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CTTCTCCACAGACCCAGAGGT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. maxima F3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*ACTACGGAAAAGTGCGTAGCT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. maxima B3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CCTTCCTCCCTTCTGAAAACTG</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. maxima FIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GAGTCACTGCTGATGTACCAAA 
			AG-GAACTATGCCGCTTTCCCCTG</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. maxima BIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*AGAATGCGGATTTGTTAGCAGC-AGCAAGTCCAAGGTGTGTGTA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. maxima LB</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CAAGCCTACGCGGACATC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. maxima LF</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*TTATGCAGCTGGGTCAACG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. mitis F3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*ACGATAGCCAAGACACGTAAGG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. mitis B3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CCCCGTGATAAGAGTAGGAACA</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. mitis FIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CGCGGGTCGTGAGATTTAAATT 
			AT-GGAAGATCAGGACGGGCACT</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. mitis BIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GTTTCAGTTGATGAACAAGCGA 
			GA-TGCGCCTCTAGAATCAAGACG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. mitis LB</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*TCCATGCATCCCCTTGTT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. mitis LF</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CGTGGGCACAGATTGATTC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. necatrix F3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*TGGCTTTCCCGCGTACC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. necatrix B3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CGGCCCAACACAAAGACTG</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. necatrix FIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CGCTTGAGTTTTAAGCTATGCA 
			CA-GACCCAAGCAGCTCACCAA</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. necatrix BIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CGCCATGCCATTCAATGAACG-*GAGGCATACCGGCGTTGTC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. necatrix LB</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GTCTGTAACTTGGGACGTTGT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. necatrix LF</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GAACAGCCGGAGCCTCTC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. praecox F3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GCCCTTGTATGTTGCTGTTTCT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. praecox B3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GCGCACGAATCTGAATCACAC</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. praecox FIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*ATCTCCTCAAAGACTTTCGCGT 
			A-GCGCTTGGCTATATCCATAGG</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. praecox BIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GCTCTCGTGGCATACTTGC-GCCAGGAGCCACTGATTGT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. praecox LB</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GAATAGCATTGCCAGGTGG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. praecox LF</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GTCCACTGTCATTAATATTGC 
			TGC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. tenella F3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GCTTGTGAAGGTCAGCGTG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. tenella B3</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GCTGAGTCCATACGTACTTCCT</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. tenella FIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GCCACTGCTATGGAAAGTCAC 
			AC-CATAACTGGCATGCAGGGGT</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. tenella BIP</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*GTTTGGCCCGAAAGTTGTGAA 
			GA-CGTCAGAAATTGCTGCCCAAT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">E. tenella LB</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CGCATGTGCAGTTGAAGACA</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">E. tenella LF</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">VC00021</td>
			<td style="width:295px;">*CCAAATGTATCTGCTAGTTATA 
			TTAACAAG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">10 x ThermoPol reaction buffer</td>
			<td style="width:161px;">New England Biolabs</td>
			<td style="width:139px;">B9004S</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:295px;">MgSO4</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">M7506</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">dNTPs</td>
			<td style="width:161px;">Promega</td>
			<td style="width:139px;">U1330</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Betaine solution (5 M)</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">B0300</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Bst polymerase</td>
			<td style="width:161px;">New England Biolabs</td>
			<td style="width:139px;">M0275S</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Hydroxynaphthol blue</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:139px;">33936</td>
			<td style="width:295px;">Dissolved in molecular grade water.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">UltraPure agarose</td>
			<td style="width:161px;">Invitrogen</td>
			<td style="width:139px;">16500-500</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">10 x Tris/Borate/EDTA (TBE) buffer</td>
			<td style="width:161px;">Invitrogen</td>
			<td style="width:139px;">AM9863</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Blue/Orange DNA loading dye (x6)</td>
			<td style="width:161px;">Promega</td>
			<td style="width:139px;">G1881</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">GeneRuler 1Kb DNA ladder</td>
			<td style="width:161px;">Thermo Scientific</td>
			<td style="width:139px;">SM0313</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">SafeView nucleic acid stain</td>
			<td style="width:161px;">NBS Biologicals</td>
			<td style="width:139px;">NBS-SV</td>
			<td style="width:295px;"></td>
		</tr>
	</tbody>
</table>

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.

アッセイの検証検証中に各アイメリア種特異的LAMPアッセイは、ホスト制御ニワトリ、ならびにニワトリのゲノムDNAを感染させる7つ全てのアイメリア種を表す純粋なDNAサンプルのパネルを用いて試験した。アガロースゲル電気泳動は、各アッセイを解決するために使用しないホスト交差反応性18絶対種特異性を実証した。次に、10倍連続希釈系列は、精製されたニワトリ盲腸コクシジウムゲノムDNAを使用して調製18 1〜10ゲノムコピーのアッセイの感度限界を明らかにした。上限は最高濃度（100,000ゲノムコピー）18を含む正まで達成された結果として決定しなかった。 フィールドサンプルを使用したアプリケーション アイメリア試験のために収集されたサンプルは、pの結果として淘汰死んでいるのが見つかったニワトリに由来する可能性が高いOOR健康または1〜3の可能性が高いサンプルサイズを示す、センチネル健康監視のために淘汰時ルーチンの一部。サーベイランスプログラムの一環として、米国のブロイラー農場から収集三鳥のテストは、腸のサンプルを3組が得られた。各アイメリア種 （ 表1）のための好ましい腸の部位に優先順位を、種特異的LAMPアッセイのアプリケーションをターゲットと、指示薬として青色hydroxynaphthol（ 図2A）を使用して試験したすべての鳥eimerian感染の視覚的識別を可能にした。 hydroxynaphtholブルーを使用した場合、負のLAMP反応を達成色がピンクに紫の範囲であるが、常に正の結果によって達成青と区別されることができる。アガロースゲル電気泳動による確認は、比較結果（ 図2B）を得た。電界印加時にはユーザーが全7種に対してフルスクリーンを適用することを選択、またはとしての優先順位のみ種を標的にすることができる重要またはファームまたは周辺地域に循環していることが知られ。 アイメリアの発生のための診断法として確立するためのPCRベースのアプローチの失敗は、新しいテストを簡単にするための要件を強調している。 LAMPは、PCRよりも簡単準備と処理を提供していますが、鳥ごとに複数の腸のサイトをテストするための要件は、落胆のまま。 1つ以上のLAMPアッセイを用いて試験することができる鳥ごとに単一のプールされたDNA試料の製造は、より魅力的である可能性が高い。すべての7つのLAMPアッセイでテストするために、表1に記載した前のDNA調製物をプール特定の腸の部位のそれぞれから収集材料を表す鳥ごとに1つのプールされたサンプルを処理して各腸部位が別々に処理された（ 図2と同じ結果を提供図3）と比較した。 <img alt="図1" src=「/files/ftp_upload/52552/52552fig1.jpg &quot;/&gt; 鶏に感染するアイメリア種の寄生虫のLAMP検出のための図1.腸サンプリングサイトは。各アイメリア種の標的と腸の領域は、E。（点線黒線の間の数で示されたサンプリングの好ましい部位で、色付きの線で強調表示されているアセルブリナ ：黄色/ 1、 イー·ブルネッティ ：ピンク/ 2、E。マキシマ ：ブルー/ 3、 イー·ミティス ：赤/ 5、E.早発 ：オレンジ/ 4、 大腸菌necatrixグリーン/ 6およびE.テネラ ：グレー/ 7）。 <a href="https://www.jove.com/files/ftp_upload/52552/52552fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/52552/52552fig2.jpg" /> あちこちeimerian感染の図2. LAMP診断mは3種の市販のブロイラー鶏。LAMP反応は（A）hydroxynaphtholブルーを使用して解決、水色反応が陽性であったピンクの反応に紫色が負であり、（B）アガロースゲル電気泳動。各寄生種は、表1に示すように、サンプリングされた腸の部位であった。 = E.アセルブリナ 、B = E。ブルネッティ 、馬= E.マキシマ 、ミ= E.ミチス 、N = E. necatrix、P = E.早発とT = E.テネラ 。レーン1は、GeneRuler 1KbのDNAラダーを含んでいた。 <a href="https://www.jove.com/files/ftp_upload/52552/52552fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/52552/52552fig3.jpg" /> 3つの別々のコマーシャルブロイラーからプールされたサンプルを使用してeimerian感染の図3. LAMP診断。LAMPのREACンはピンクの反応にhydroxynaphtholスカイブルーの反応が陽性であった青、紫を使用して解決陰性であった。 = E.アセルブリナ 、B = E。ブルネッティ 、馬= E.マキシマ 、ミ= E.ミチス 、N = E. necatrix、P = E.早発とT = E.テネラ 。 <a href="https://www.jove.com/files/ftp_upload/52552/52552fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> サンプルサイト </td><td> アイメリア種アッセイ（最も可能性が高い） </td></tr><tr><td>十二指腸（D） </td><td> E.アセルブリナ、E。早発 </td></tr><tr><td>空腸/回腸*（J / I） </td><td> E.マキシマ、E。necatrix </td></tr><tr><td>盲腸（C） </td><td> 大腸菌necatrix、E。テネラ </td></tr><tr><td>回腸末端（TI） </td><td> E.ブルネッティ、E。ミチス </td></tR&gt; <tr><td>プールされた試料（P） </td><td> E.アセルブリナ、E。ブルネッティ、E。マキシマ、E。ミチス、大腸菌necatrix、E。早発、E.テネラ </td></tr></tbody></table> 候補アイメリア種アッセイの表1.腸地域固有の選択。図1に示すように、サンプリングされる領域の選択は、各アイメリア種によって異なります。プールされたサンプルは、その後DNA調製のために組み合わされたすべての4つの特定のサイトから収集した材料を含む。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> プライマー* </td><td> ストック濃度（μM） </td><td> 容量（μL） </td></tr><tr><td>水</td><td> - </td><td> 60 </td></tr><tr><td>フォワードインナープライマー（FIP） </td><td> 100 </td><td> 40 </td></tr><tr><td>後方インナーぷりメール（BIP） </td><td> 100 </td><td> 40 </td></tr><tr><td>フォワードアウタープライマー（F3） </td><td> 100 </td><td> 10 </td></tr><tr><td>後方外側プライマー（B3） </td><td> 100 </td><td> 10 </td></tr><tr><td>ループフォワード（LF） </td><td> 100 </td><td> 20 </td></tr><tr><td>後方ループ（LB） </td><td> 100 </td><td> 20 </td></tr><tr><td> 合計 </td><td></td><td> 200 </td></tr></tbody></table> LAMPプライマープレミックスの表2.準備。LAMP用プライマーミックスを準備するために必要なコンポーネントとの比率。示さボリュームは100 LAMP反応のためである。材料とBarkway ら （2011）18に示すように*プライマーアイデンティティ。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> 株式の濃nを </td><td> 最終的な反応を濃nを </td><td> 反応あたり容量（μL） </td></tr><tr><td> DDW </td><td> - </td><td> - </td><td> 10.1 </td></tr><tr><td> ThermoPolバッファ</td><td> 10× </td><td> 1× </td><td> 2.5 </td></tr><tr><td> MgSO 4を</td><td> 100 mMの</td><td> 2 mMの</td><td> 0.5 </td></tr><tr><td>プライマーミックス* </td><td colspan="2">表2 </td><td> 2.5 </td></tr><tr><td>のdNTP </td><td> 25 mMの</td><td> 400μmの</td><td> 0.4 </td></tr><tr><td>ベタイン</td><td> 5 M </td><td> 1 M </td><td> 5 </td></tr><tr><td> Hydroxynaphtholブルー</td><td> 3 mMの</td><td> 120μM </td><td> 1 </td></tr><tr><td> Bst DNAポリメラーゼ</td><td> 8000 U / mlの</td><td> 8 U </td><td> 1 </td></tr><tr><td> 合計 </td><td></td><td></td><td> 23 </td></tr></tbody></table> LAMの表3.準備P反応マスターミックス。* アイメリア種特異的。

Watch this Scientific Journal Video about Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens at JoVE.com

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 ...

loop-mediated-isothermal-amplification-lamp-assays-for-species

Research

JoVE Journal

Immunology and Infection

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

26.8K ビュー.  Royal Veterinary College, London. 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.

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

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

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

標準的な臨床アッセイによる検出限界以下のウイルス量とHIV感染個体におけるHIV - 1 RNAを増幅して定量化

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

タングウイルスに対する新規抗ウイルス剤の同定のためのアッセイ

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

感染するためのプロトコル<em&gt;線虫（Caenorhabditis elegans）</em&gt;と<em&gt;ネズミチフス菌</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

抗ウイルス化合物の同定および評価のための早期のウイルス侵入アッセイ

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性的および定量的検出のためのアッセイおよびタンパク質の抗菌薬のキャラクタリゼーション

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

の検出のための凍結乾燥LAMP法試薬の開発<em&gt; Q熱リケッチア</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

細菌の付着と血清抵抗性ビトロネクチンの役割を研究するための試金

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug&#39;s pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples.
In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-na&#239;ve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

Here we present a cell-based flow cytometry method to detect neutralizing antibodies or other factors that interfere with the cellular uptake of enzyme replacement therapies in a human matrix, such as cerebral spinal fluid (CSF) or human serum.

細胞に基づく試金の酵素補充療法の細胞内取り込みを中和する抗体の検出

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The ...

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 ...