この研究は、グローバル·ヘルス·イニシアティブにおけるグランド課題を通してビル·アンド·メリンダ·ゲイツ財団からの助成金によって賄われていた。

<ol>
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F., von Nickisch-Rosenegk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+Plasmodium+falciparum+with+isothermal+recombinase+polymerase+amplification+and+lateral+flow+analysis.">Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis.</a> Malaria Journal. 13 (1), 99 (2014).</li><li>Crannell, Z. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+Acid+test+to+diagnose+cryptosporidiosis:+lab+assessment+in+animal+and+patient+specimens.">Nucleic Acid test to diagnose cryptosporidiosis: lab assessment in animal and patient specimens.</a> Analytical Chemistry. 86 (5), 2565-2571 (2014).</li><li>Rohrman, B. A., Richards-Kortum, R. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T.+gondii+RP+Promoters+&amp;+Knockdown+Reveal+Molecular+Pathways+Associated+with+Proliferation+and+Cell-Cycle+Arrest.">T. gondii RP Promoters &amp; Knockdown Reveal Molecular Pathways Associated with Proliferation and Cell-Cycle Arrest.</a> PLoS ONE. 5 (11), 15 (2010).</li><li>Segall, H. I., Yoo, E., Sutton, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+detection+of+artificial+replication-competent+lentivirus+of+altered+host+range.">Characterization and detection of artificial replication-competent lentivirus of altered host range.</a> Molecular Therapy. 8 (1), 118-129 (2003).</li><li>Crannell, Z. A., Rohrman, B. 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The authors declare they have no competing financial interests.

プロンプトが表示されたら、MATLABアルゴリズムを使用して、意味のある定量データを得るためには、ユーザは、適切な入力値を選択しなければならない。セクション5および6の各スクリプトを開始した後、すべての入力変数は自動的にコマンドウィンドウに要請され、出力は自動的に生成される。 5.7節では、ユーザーは勾配閾値を選択するよう求めている。勾配閾値の値は、適合の相関係数?...

定量的核酸増幅は、環境、食品媒介性および水媒介性病原体の検出のためだけでなく、臨床診断のための重要な技術である。リアルタイム定量的ポリメラーゼ連鎖反応（qPCR）核酸の感度が高く、特異的かつ定量的検出のための金の標準的な方法で、 例えば、HIV-1ウイルス負荷試験、細菌性病原体の検出、および他の多くの生物の1のスクリーニングのための- 3。リアルタイム定量PCRの間に、プライマーは、サイクル中の病原体のDNAを増幅し、そして蛍光シグナルは各サイクルにおいて、サンプル中の増幅されたDNAの量に比例して生成される。病原体DNAの未知の濃度を含有する試料は、標準試料の初期DNA濃度と蛍光シグナルが特定の閾値（ すなわち、サイクル閾値、またはC T）に達した時点に関する標準曲線を用いて定量することができる。 、4開発されている。これらのプラットフォームは、一般に、より迅速な結果を提供し、より安価な、より簡単な装置を用いて達成され得るより低い、単一温度で核酸を増幅する。 、ポイントオブケア用途に特に魅力的である分でDNAを増幅するRPAは、低い増幅温度（37℃）を必要とし、不純物5,6の存在下で活性なままである。 12 - RPAアッセイbiothreat剤7の食品分析、病原体検出、癌の薬物スクリーニング、および検出を含む、広範囲の用途のために開発されている。しかし、核酸の定量化のためのRPAの使用が制限されてきた13,14。 前作では、翔だったwnは、リアルタイム定量的RPA（QRPA） を15可能である。ここで、より詳細なプロトコルは、標準曲線、定量PCRを用いて定量化に類似する方法を用いて未知のサンプルを定量するためにリアルタイム定量RPAを使用するために提供される。このプロトコルは、同様に、システムが適切に機能していることを確認するために内部陽性対照（IPC）を開発する方法のように、概念実証としてHIV-1 DNAを検出するために、サーマルサイクラーにRPA反応を実行する方法について説明します。トレーニングデータを使用して標準曲線を構築するためのサーマルサイクラーや顕微鏡とデータ解析を使用して、データ収集にも詳述されている。最後に、カスタムスクリプトを使用して標準曲線を使用して、未知のサンプルを定量するための方法が示されている。このQRPA技術は、未知の濃度のサンプルの定量を可能にし、伝統的なリアルタイム定量PCRに比べて多くの利点を有する。

<table border="0" cellpadding="0" cellspacing="0" width="1426">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">qRPA Assay</td>
			<td style="width:199px;"></td>
			<td style="width:195px;"></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Supply</td>
			<td>Vendor</td>
			<td>Part number</td>
			<td style="width:589px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HIV-1 forward primer</td>
			<td>Integrated DNA Technologies</td>
			<td>custom DNA oligos</td>
			<td style="width:589px;">5&#8217;-TGG CAG TAT TCA TTC ACA ATT TTA AAA GAA AAG G-3&#8217;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HIV-1 reverse primer</td>
			<td>Integrated DNA Technologies</td>
			<td>custom DNA oligos</td>
			<td style="width:589px;">5&#8217;-CCC GAA AAT TTT GAA TTT TTG TAA TTT GTT TTT G-3&#8217;&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HIV-1 probe</td>
			<td>BioSearch Technologies&#160;</td>
			<td>custom DNA oligos</td>
			<td style="width:589px;">5&#8217;- TGC TAT TAT GTC TAC TAT TCT TTC CCC [SIMA/HEX] GC [THF] C [dT-BHQ1] GTA CCC CCC AAT CCC C -3&#8217;&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IPC probe</td>
			<td>BioSearch Technologies&#160;</td>
			<td>custom DNA oligos</td>
			<td style="width:589px;">5&#8217;-AGG TAG TGA CAA GAA ATA ACA ATA CAG GAC [FAM] T [THF] T [dT-BHQ1] GGT TTT GTA ATT GGA A -3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPA exo reagents (pellets, rehydration buffer, magnesium acetate</td>
			<td>TwistDx</td>
			<td>TwistAmp exo</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR tube strips</td>
			<td>BioRad</td>
			<td>TLS0801</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR flat cap tube strips</td>
			<td>BioRad</td>
			<td>TCS0803</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro-seal adhesive</td>
			<td>BioRad</td>
			<td>558/MJ&#160;</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">HIV-1 target (pHIV-IRES- eYFP&#916;Env&#916;Vif&#916;Vpr)</td>
			<td></td>
			<td></td>
			<td style="width:589px;">custom plasmid, see: Segall, H. I., Yoo, E. &#38; Sutton, R. E. Characterization and detection of artificial replication-competent lentivirus of altered host range. Molecular Therapy 8, 118&#8211;129, doi:10.1016/S1525-0016(03)00134-5 (2003).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human male genomic DNA</td>
			<td>Applied Biosystems</td>
			<td>360486</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well cold-block</td>
			<td>Cole Parmer</td>
			<td>EW-36700-48</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermal cycler</td>
			<td>BioRad</td>
			<td>CFX96</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MiniFuge</td>
			<td>VWR</td>
			<td>93000-196</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vortex</td>
			<td>VWR</td>
			<td>58816-121</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris buffer 1.0 M, pH 8.0</td>
			<td>EMD Millipore</td>
			<td>648314</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA 0.5 M, pH 8.0</td>
			<td>Promega</td>
			<td>V4321</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclease free water</td>
			<td>Ambion</td>
			<td>AM9937</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IPC Development</td>
			<td></td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Supply</td>
			<td>Vendor</td>
			<td>Part number</td>
			<td style="width:589px;">Comments/Description</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Cryptosporidium parvum IPC template</td>
			<td>Waterborne Inc</td>
			<td>P102C</td>
			<td style="width:589px;">It is also possible to order a double stranded synthetic target from IDT if the user is unequipped to work with C. parvum (a BSL-2 infectious agent). PCR and RPA primers for C. parvum were designed using GenBank accession number AF115377.1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR long forward primer</td>
			<td>Integrated DNA Technologies</td>
			<td>custom DNA oligos</td>
			<td>5&#8217;-TGG CAG TAT TCA TTC ACA ATT TTA AAA GAA AAG G/ ATC TAA GGA AGG CAG CAG GC-3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR long reverse primer</td>
			<td>Integrated DNA Technologies</td>
			<td>custom DNA oligos</td>
			<td>5&#8217;- CCC GAA AAT TTT GAA TTT TTG TAA TTT GTT TTT G/ TGC TGG AGT ATT CAA GGC ATA -3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phusion High-Fidelty DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0530S</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Qiaquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TAE 10X buffer</td>
			<td>EMD Millipore</td>
			<td>574797</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose</td>
			<td>Sigma Aldrich</td>
			<td>A9539</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope Experiments</td>
			<td></td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Supply</td>
			<td>Vendor</td>
			<td>Part number</td>
			<td style="width:589px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Upright fluorescence microscope</td>
			<td>Zeiss</td>
			<td>Zeiss Imager.J1</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stage heater</td>
			<td>Bioscience Tools</td>
			<td>TC-GSH</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Channel Precision High Stability Temperature Controller</td>
			<td>Bioscience Tools</td>
			<td>TC-1100S</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FAM/GFP filter cube</td>
			<td>Zeiss</td>
			<td>filter set 38 (000000-1031-346)</td>
			<td style="width:589px;">excitation BP 470/40 nm, emission BP 520/50 nm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEX filter cube</td>
			<td>Chroma</td>
			<td>49014</td>
			<td style="width:589px;">excitation BP 530/30 nm, emission BP 575/40 nm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laser cutter</td>
			<td>Engraver&#39;s Network</td>
			<td>VLS3.60</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1/8&#34; black acrylic</td>
			<td>McMaster Carr</td>
			<td>8505K113</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 mm clear acrylic</td>
			<td>McMaster Carr</td>
			<td>PD-72268940&#160;</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Super glue</td>
			<td>Office Depot</td>
			<td>Duro super glue&#160;</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR grade mineral oil</td>
			<td>Sigma Aldrich</td>
			<td>M8662-5VL</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Data Analysis</td>
			<td></td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software</td>
			<td>Vendor</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB</td>
			<td>MATLAB</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB script: &#34;JoVE_qRPA_standard_curve.m&#8221;</td>
			<td>Included in SI</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB script: &#34;JoVE_qRPA_validation_and_quantification.m&#8221;</td>
			<td>Included in SI</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB script: &#34;JoVE_real_time_intensity_to_excel.m&#8221;</td>
			<td>Included in SI</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adobe Illustrator</td>
			<td>Adobe</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">JoVE_qRPA_well.ai</td>
			<td>Included in SI</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">JoVE_qRPA_base.ai</td>
			<td>Included in SI</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AxioVision software</td>
			<td>Zeiss</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">JoVE_AxioVision_Script.ziscript</td>
			<td>Included in SI</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
	</tbody>
</table>

development of a quantitative recombinase polymerase amplification assay with an internal positive control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

ターゲットとQRPA実験でIPCとして機能する配列を選択する前に（HIV-1）DNA、内部陽性対照（IPC）の候補が生成され、HIV-1 DNAの存在なしQRPA反応において増幅するそれらの能力についてスクリーニングした。 IPC候補は、アウト競合HIV-1アンプリコンの形成からIPC形成を防止するために、ターゲット（HIV-1）DNAよりも長い。 図2Aは 、2℃の生成に示すようにパルバム IPC候補?...

Watch this Scientific Journal Video about Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control at JoVE.com

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

内部陽性対照との定量的リコンビナーゼポリメラーゼ増幅アッセイの開発

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...

development-quantitative-recombinase-polymerase-amplification-assay

Research

JoVE Journal

Biology

13.4K ビュー.  Rice University. Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.