이 연구는 세계 보건 이니셔티브에있는 그랜드 도전을 통해 빌 &amp; 멜린다 게이츠 재단의 연구비 지원을 받았다.

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+detection+using+recombination+proteins.">DNA detection using recombination proteins.</a> PLoS Biology. 4 (7), 1115-1121 (2006).</li><li>Kr&#245;lov, K., Frolova, J., 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=Sensitive+and+Rapid+Detection+of+Chlamydia+trachomatis+by+Recombinase+Polymerase+Amplification+Directly+from+Urine+Samples.">Sensitive and Rapid Detection of Chlamydia trachomatis by Recombinase Polymerase Amplification Directly from Urine Samples.</a> The Journal of Molecular Diagnostics JMD. 16 (1), 127-135 (2014).</li><li>Santiago-Felipe, S., Tortajada-Genaro, L. a., Puchades, R., Maquieira, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinase+polymerase+and+enzyme-linked+immunosorbent+assay+as+a+DNA+amplification-detection+strategy+for+food+analysis.">Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis.</a> Analytica Chimica Acta. 811 (1), 81-87 (2014).</li><li>Kersting, S., Rausch, V., Bier, F. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+paper+and+plastic+device+for+performing+recombinase+polymerase+amplification+of+HIV+DNA.">A paper and plastic device for performing recombinase polymerase amplification of HIV DNA.</a> Lab on a Chip. 12 (17), 3082 (2012).</li><li>Loo, J. F. C., Lau, P. M., Ho, H. P., Kong, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+aptamer-based+bio-barcode+assay+with+isothermal+recombinase+polymerase+amplification+for+cytochrome-c+detection+and+anti-cancer+drug+screening.">An aptamer-based bio-barcode assay with isothermal recombinase polymerase amplification for cytochrome-c detection and anti-cancer drug screening.</a> Talanta. 115 (1), 159-165 (2013).</li><li>Euler, M., 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=Development+of+a+panel+of+recombinase+polymerase+amplification+assays+for+detection+of+biothreat+agents.">Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents.</a> Journal of Clinical Microbiology. 51 (4), 1110-1117 (2013).</li><li>Abd El Wahed, 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=A+portable+reverse+transcription+recombinase+polymerase+amplification+assay+for+rapid+detection+of+foot-and-mouth+disease+virus.">A portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus.</a> PloS One. 8 (8), e71642 (2013).</li><li>Escadafal, C., 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=Rapid+Molecular+Assays+for+the+Detection+of+Yellow+Fever+Virus+in+Low-Resource+Settings.">Rapid Molecular Assays for the Detection of Yellow Fever Virus in Low-Resource Settings.</a> PLoS Neglected Tropical Diseases. 8 (3), e2730 (2014).</li><li>Hutson, S. 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. A., Richards-Kortum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+HIV-1+DNA+using+Real-Time+Recombinase+Polymerase+Amplification.">Quantification of HIV-1 DNA using Real-Time Recombinase Polymerase Amplification.</a> Analytical Chemistry. 86 (12), (2014).</li><li>Sollis, K. 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=Systematic+review+of+the+performance+of+HIV+viral+load+technologies+on+plasma+samples.">Systematic review of the performance of HIV viral load technologies on plasma samples.</a> PloS one. 9 (2), e85869 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. , 1-166 (2011).</li></ol>

The authors declare they have no competing financial interests.

프롬프트되면 MATLAB 알고리즘을 사용하여 의미있는 정량화 된 데이터를 획득하기 위해, 사용자는 해당 입력 값을 선택한다. 제 5,6의 각 스크립트를 개시 한 후, 모든 입력 변수는 자동 명령 창에서 요청 된 것으로 출력이 자동으로 생성된다. 5.7 절에서는 사용자가 기울기 임계 값을 선택하라는 메시지가 표시됩니다. 경사의 문턱 값은 착용감 상관 계수 (R 2)의 제곱에 영향을 미친다. 열 사?...

정량적 핵산 증폭 환경, 인성 및 인성 병원균의 검출뿐만 아니라, 임상 적 진단을위한 중요한 기술이다. 실시간 정량적 폴리머 라제 연쇄 반응 (qPCR에)는 HIV-1 바이러스 성 부하 테스트 세균성 병원체의 검출 및 다른 많은 생물 한 스크리닝 예를 들면 핵산의 민감하고 특이 및 정량적 검출 금 표준 방법 - 3. 실시간 qPCR의 동안 프라이머 사이클 병원체 DNA를 증폭 및 형광 신호는 각주기에서 증폭 된 DNA 시료의 양에 비례한다고 생성된다. 병원체 DNA의 미지 농도를 함유하는 샘플은 표준 시료의 초기 DNA 농도와 형광 신호가 특정 임계 값에 도달하는 시간 (즉, 사이클 임계치 또는 C의 T)에 관한 표준 곡선을 사용하여 정량화 될 수있다. 4, 개발되어왔다. 이러한 플랫폼은 일반적으로 더 빠른 결과를 제공하고 덜 비싼, 간단한 장치로 달성 될 수있는 저급 단일 온도에서 핵산을 증폭. , 현장 진료 응용 프로그램에 특히 매력적인 분 안에 DNA를 증폭 RPA는 낮은 증폭 온도 (37 ° C)를 필요로하고, 불순물 5,6의 존재에 활성 상태로 유지됩니다. 12 - RPA 분석법은 biothreat 제 7의 식품 분석, 병원균 검출, 항암제 스크리닝 및 검출을 포함한 다양한 애플리케이션을 위해 개발되었다. 그러나, 핵산의 정량 RPA를 사용 13,14 한정되었다. 이전의 연구에서는 웃음이었다WN 그 실시간 정량적 RPA (qRPA)는 15 가능하다. 여기서, 상세한 프로토콜은 표준 곡선을 사용하여 정량화 qPCR에 유사 방법을 사용하여 미지 시료를 정량화 실시간 정량적 RPA를 사용하기 위해 제공된다. 이 프로토콜은 제대로 작동하는 시스템을 보장하기 위해 내부 양성 대조군 (IPC)를 개발하는 방법뿐만 아니라, 개념 증명으로 HIV-1 DNA를 검출하기 위해 열 자전거 타는 사람에 RPA 반응을 수행하는 방법에 대해 설명합니다. 트레이닝 데이터를 이용하여 표준 곡선을 구성하기위한 서열 증폭기 또는 현미경과 데이터 분석에 의한 데이터 수집은 또한 상술된다. 마지막으로, 커스텀 스크립트 표준 곡선을 사용하여 미지 시료 정량법이 설명된다. 이 qRPA 기술은 미지 농도와 시료의 정량 분석​​을 가능하게하고 기존의 실시간 qPCR에 비해 많은 장점이 있습니다.

<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.

목표 qRPA 실험에서 IPC 역할을하는 시퀀스를 선택하기 전에 (HIV-1) DNA, 내부 양성 대조군 (IPC) 후보가 생성되어 HIV-1 DNA가없는 상태 qRPA 반응으로 증폭 할 수있는 능력에 대한 검사. IPC 후보 타겟 (HIV-1)의 아웃 - 경쟁 HIV-1 앰플 리콘 형성에서 IPC 형성되지 않도록 DNA보다 길다. 도 2a, C.의 두 세대에 도시 된 바와 같이 IPC 포자충 후보들 겔 전기 영동을 사용하여 415 및 435 bp의 밴?...

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.

내부 양성 대조군과 양적 재조합 효소 중합 효소 증폭 분석 개발

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 조회수.  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.

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

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

특정 사이트 Recombinase (SSR) 기술의 원리

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

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생물학

Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase

HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies primarily target virus enzymes or virus-cell fusion, suppressing the viral life cycle without eradicating the infection. Since the integrated provirus is not targeted by these approaches, new resistant strains of HIV-1 may emerge. Here, we report that the engineered recombinase Tre (see Molecular evolution of the Tre recombinase , Buchholz, F., Max Planck Institute for Cell Biology and Genetics, Dresden) efficiently excises integrated HIV-1 proviral DNA from the genome of infected cells. We produced loxLTR containing viral pseudotypes and infected HeLa cells to examine whether Tre recombinase can excise the provirus from the genome of HIV-1 infected human cells. A virus particle-releasing cell line was cloned and transfected with a plasmid expressing Tre or with a parental control vector. Recombinase activity and virus production were monitored. All assays demonstrated the efficient deletion of the provirus from infected cells without visible cytotoxic effects. These results serve as proof of principle that it is possible to evolve a recombinase to specifically target an HIV-1 LTR and that this recombinase is capable of excising the HIV-1 provirus from the genome of HIV-1-infected human cells.
Before an engineered recombinase could enter the therapeutic arena, however, significant obstacles need to be overcome. Among the most critical issues, that we face, are an efficient and safe delivery to targeted cells and the absence of side effects.

Current HIV-1 strategies act to suppress the viral life cycle but do not effectively eradicate infection. Here, we demonstrate that an engineered recombinase can efficiently excise integrated HIV-1 proviral DNA from the genome of infected cells.

인터뷰 : 진화 Recombinase을 사용하여 HIV - 1의 DNA 절단 Proviral

HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Neurotoxicity 높은 컨텐츠 분석의 연결 및 Astrocyte 공동 문화 분석

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

인슐린을 표현 콜로니를 형성 Progenitors에 대한 정량 분석

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

주름 식민지 개발을 사용하여 Biofilm 형성을 평가하기 위해 세미 양적 접근

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

멀티 플렉스 항체 검사 방법을 사용하여 Luminex xMAP 분석에 캡처 엘리사의 전환

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

정량 면역 형광 분석은 중심체에서 단백질 수준의 변화를 측정하기 위해

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

다중 파라미터 관류 조절을 통한 원발성 쥐 간세포의 분리

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

Application of Ultrasound and Shear Wave Elastography Imaging in a Rat Model of NAFLD/NASH

Nonalcoholic Steatohepatitis (NASH) is a condition within the spectrum of Non-Alcoholic Fatty Liver Disease (NAFLD), which is characterized by liver fat accumulation (steatosis) and inflammation leading to fibrosis. Preclinical models closely recapitulating human NASH/NAFLD are essential in drug development. While liver biopsy is currently the gold standard for measuring NAFLD/NASH progression and diagnosis in the clinic, in the preclinical space, either collection of whole liver samples at multiple timepoints during a study or biopsy of liver is needed for histological analysis to assess the disease stage.
Conducting a liver biopsy mid-study is an invasive and labor-intensive procedure, and collecting liver samples to assess disease level increases the number of research animals needed for a study. Thus, there is a need for a reliable, translatable, non-invasive imaging biomarker to detect NASH/NAFLD in these preclinical models. Non-invasive ultrasound-based B-mode images and Shear Wave Elastography (SWE) can be used to measure steatosis as well as liver fibrosis. To assess the utility of SWE in preclinical rodent models of NASH, animals were placed on a pro-NASH diet&#160;and underwent non-invasive ultrasound B-mode and shear wave elastography imaging to measure hepatorenal (HR) index and liver elasticity, measuring progression of both liver fat accumulation and tissue stiffness, respectively, at multiple time points over the course of a given NAFLD/NASH study.
The HR index and elasticity numbers were compared to histological markers of steatosis and fibrosis. The results showed strong correlation between the HR index and percentage of Oil Red O (ORO) staining, as well as between elasticity and Picro-Sirius Red (PSR) staining of livers. The strong correlation between classic ex vivo methods and in vivo imaging results provides evidence that shear wave elastography/ultrasound-based imaging can be used to assess disease phenotype and progression in a preclinical model of NAFLD/NASH.

This protocol describes the use of an enhanced ultrasound technique to non-invasively observe and quantify liver tissue changes in rodent models of nonalcoholic fatty liver disease.

NAFLD/NASH의 쥐 모형에 초음파 및 전단 파 단층 사진 의 응용

Nonalcoholic Steatohepatitis (NASH) is a condition within the spectrum of Non-Alcoholic Fatty Liver Disease (NAFLD), which is characterized by liver fat ...

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.