Name: development of a quantitative recombinase polymerase amplification assay with an internal positive control
Uploaded: 2015-03-30T15:00:00
Description: Watch this Scientific Journal Video about Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control at JoVE.com

This research was funded by a grant from the Bill &#38; Melinda Gates Foundation through the Grand Challenges in Global Health Initiative.

1. Program the Thermal Cycler for Real-time qRPA Reactions
<ol>
	<li>Create a new protocol in the thermal cycler software.
	<ol>
		<li>Insert a pre-incubation step: 39 &#176;C for 1 min.</li>
		<li>Add a second step: 39 &#176;C for 20 sec followed by a plate read.</li>
		<li>Finally, insert a &#8220;GO TO&#8221; that repeats the second step 59 more times.</li>
		<li>Save the protocol.</li>
	</ol>
	</li>
	<li>Create a new plate in the &#8220;plate editor&#8221; tab of the software. Select wells on the plate correspondin...

<ol>
	<li>Yoon, J. -. Y., Kim, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab-on-a-Chip+Pathogen+Sensors+for+Food+Safety.">Lab-on-a-Chip Pathogen Sensors for Food Safety.</a> Sensors. 12 (8), 10713-10741 (2012).</li><li>Quintero-Betancourt, W., Peele, E. R., Rose, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptosporidium+parvum+and+Cyclospora+cayetanensis:+A+review+of+laboratory+methods+for+detection+of+these+waterborne+parasites.">Cryptosporidium parvum and Cyclospora cayetanensis: A review of laboratory methods for detection of these waterborne parasites.</a> Journal of Microbiological Methods. 49 (3), 209-224 (2002).</li><li>Sedlak, R. H., Jerome, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+diagnostics+in+the+era+of+digital+polymerase+chain+reaction.">Viral diagnostics in the era of digital polymerase chain reaction.</a> Diagnostic Microbiology and Infectious Disease. 75 (1), 1-4 (2013).</li><li>Asiello, P. J., Baeumner, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+isothermal+nucleic+acid+amplification,+a+review.">Miniaturized isothermal nucleic acid amplification, a review.</a> Lab on a Chip. 11 (8), 1420-1430 (2011).</li><li>Piepenburg, O., Williams, C. H., Stemple, D. L., Armes, N. 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>

In order to obtain meaningful quantification data using the MATLAB algorithm, the user must select appropriate input values when prompted. After initiating each script in Sections 5 and 6, all input variables are automatically solicited in the command window and outputs are automatically generated. In Section 5.7 the user is prompted to select a slope threshold. The value of the slope threshold affects the square of the correlation coefficient (r2) of the fit. When using raw fluorescence data exported from a t...

Quantitative nucleic acid amplification is an important technique for detection of environmental, foodborne, and water-borne pathogens as well as for clinical diagnostics. Real-time quantitative polymerase chain reaction (qPCR) is the gold standard method for sensitive, specific, and quantitative detection of nucleic acids, e.g., for HIV-1 viral load testing, detection of bacterial pathogens, and screening for many other organisms1&#8211;3. During real-time qPCR, primers amplify pathogen DNA in cycles, and a fluorescent signal is generated that is proportional to the amount of amplified DNA in the sample at each cycle. A sample containing an unknown concentration of pathogen DNA may be quantified using a standard curve that relates the initial DNA concentration of standard samples and the time at which the fluorescent signal reaches a certain threshold (i.e., the cycle threshold, or CT).
Because real-time qPCR requires expensive thermal cycling equipment and several hours to receive results, alternative isothermal amplification techniques, such as recombinase polymerase amplification (RPA), have been developed4. These platforms generally provide results faster and amplify nucleic acids at a lower, single temperature, which may be accomplished with less expensive, simpler equipment. RPA, which is particularly attractive for point-of-care applications, amplifies DNA in minutes, requires a low amplification temperature (37 &#176;C), and remains active in the presence of impurities5,6. RPA assays have been developed for a wide range of applications, including food analysis, pathogen detection, cancer drug screening, and detection of biothreat agents7&#8211;12. However, use of RPA for quantification of nucleic acids has been limited13,14.
In previous work, it was shown that real-time quantitative RPA (qRPA) is feasible15. Here, a more detailed protocol is provided for using real-time quantitative RPA to quantify unknown samples using a standard curve, a method that is analogous to quantification using qPCR. This protocol describes how to perform an RPA reaction on a thermal cycler to detect HIV-1 DNA as a proof-of-concept, as well as how to develop an internal positive control (IPC) to ensure the system is functioning properly. Data collection using a thermal cycler or microscope and data analysis for constructing a standard curve using training data is also detailed. Finally, the method for quantifying unknown samples using the standard curve with a custom script is demonstrated. This qRPA technique enables quantification of samples with unknown concentrations and has many advantages over traditional real-time qPCR.

<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.

Before selecting a sequence to serve as the IPC in qRPA experiments with target (HIV-1) DNA, internal positive control (IPC) candidates are generated and screened for their ability to amplify in qRPA reactions without HIV-1 DNA present. IPC candidates are longer than the target (HIV-1) DNA to prevent IPC formation from out-competing HIV-1 amplicon formation. As shown in Figure 2A, the generation of two C. parvum IPC candidates was verified by the presence of 415 and 435 bp bands using gel electr...

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.

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

Research

JoVE Journal

Biology

Principles of Site-Specific Recombinase (SSR) Technology

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 ...

Molecular Evolution of the Tre Recombinase

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within ...

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 ...

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 ...

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 ...

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 ...

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 ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

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 ...

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 ...