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

<ol><li>Yoon, J. -Y., Kim, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lab-on-a-Chip+Pathogen+Sensors+for+Food+Safety%5BTitle%5D)+AND+%22Sensors%22%5BJournal%5D)">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/pubmed?term=((Cryptosporidium+parvum+and+Cyclospora+cayetanensis%3A+A+review+of+laboratory+methods+for+detection+of+these+waterborne+parasites%5BTitle%5D)+AND+%22Journal+of+Microbiological+Methods%22%5BJournal%5D)">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>
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<li>Asiello, P. J., Baeumner, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Miniaturized+isothermal+nucleic+acid+amplification%2C+a+review%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">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/pubmed?term=((DNA+detection+using+recombination+proteins%5BTitle%5D)+AND+%22PLoS+Biology%22%5BJournal%5D)">DNA detection using recombination proteins.</a> PLoS Biology. 4 (7), 1115-1121 (2006).</li>
<li>Krõlov, K., Frolova, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensitive+and+Rapid+Detection+of+Chlamydia+trachomatis+by+Recombinase+Polymerase+Amplification+Directly+from+Urine+Samples%5BTitle%5D)+AND+%22The+Journal+of+Molecular+Diagnostics+JMD%22%5BJournal%5D)">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/pubmed?term=((Recombinase+polymerase+and+enzyme-linked+immunosorbent+assay+as+a+DNA+amplification-detection+strategy+for+food+analysis%5BTitle%5D)+AND+%22Analytica+Chimica+Acta%22%5BJournal%5D)">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/pubmed?term=((Rapid+detection+of+Plasmodium+falciparum+with+isothermal+recombinase+polymerase+amplification+and+lateral+flow+analysis%5BTitle%5D)+AND+%22Malaria+Journal%22%5BJournal%5D)">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/pubmed?term=((Nucleic+Acid+test+to+diagnose+cryptosporidiosis%3A+lab+assessment+in+animal+and+patient+specimens%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">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/pubmed?term=((A+paper+and+plastic+device+for+performing+recombinase+polymerase+amplification+of+HIV+DNA%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">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/pubmed?term=((An+aptamer-based+bio-barcode+assay+with+isothermal+recombinase+polymerase+amplification+for+cytochrome-c+detection+and+anti-cancer+drug+screening%5BTitle%5D)+AND+%22Talanta%22%5BJournal%5D)">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/pubmed?term=((Development+of+a+panel+of+recombinase+polymerase+amplification+assays+for+detection+of+biothreat+agents%5BTitle%5D)+AND+%22Journal+of+Clinical+Microbiology%22%5BJournal%5D)">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/pubmed?term=((A+portable+reverse+transcription+recombinase+polymerase+amplification+assay+for+rapid+detection+of+foot-and-mouth+disease+virus%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">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/pubmed?term=((Rapid+Molecular+Assays+for+the+Detection+of+Yellow+Fever+Virus+in+Low-Resource+Settings%5BTitle%5D)+AND+%22PLoS+Neglected+Tropical+Diseases%22%5BJournal%5D)">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/pubmed?term=((T.+gondii+RP+Promoters+%26+Knockdown+Reveal+Molecular+Pathways+Associated+with+Proliferation+and+Cell-Cycle+Arrest%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">T. gondii RP Promoters & 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/pubmed?term=((Characterization+and+detection+of+artificial+replication-competent+lentivirus+of+altered+host+range%5BTitle%5D)+AND+%22Molecular+Therapy%22%5BJournal%5D)">Characterization and detection of artificial replication-competent lentivirus of altered host range.</a> Molecular Therapy. 8 (1), 118-129 (2003).</li>
<li>DNA Extraction from Serum. Life Technologies. , Available from:
 
 <a target="_blank" href="http://www.lifetechnologies.com/us/en/home/references/protocols/nucleic-acid-purification-and-analysis/dna-extraction-protocols/dna-extraction-from-serum.html">http://www.lifetechnologies.com/us/en/home/references/protocols/nucleic-acid-purification-and-analysis/dna-extraction-protocols/dna-extraction-from-serum.html</a> (2014).</li>
<li>Crannell, Z. A., Rohrman, B. A., Richards-Kortum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+HIV-1+DNA+using+Real-Time+Recombinase+Polymerase+Amplification%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Systematic+review+of+the+performance+of+HIV+viral+load+technologies+on+plasma+samples%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=%22Guidelines+for+the+use+of+antiretroviral+agents+in+HIV-1-infected+adults+and+adolescents%22">Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents</a>. , Department of Health and Human Services. Washington D.C. 1-166 (2011).</li>
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The authors declare they have no competing financial interests.

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 thermal cycler, values between 2.0 and 5.0 tend to yield a high r2 coefficient. In Section 5.8 the user must designate the number of standard deviations above the background to set the positive threshold. To score a sample as positive or negative, the script automatically determines the difference &#916;sample between the maximum and minimum fluorescence for each sample using the raw fluorescence data exported from the thermal cycler. It calculates the average difference &#916;background and standard deviation &#963;background for all no-target control samples. A sample is considered positive if &#916;sample is more than z &#215; &#963;background above &#916;background. In Section 5.10 the user decides whether to use the default threshold or set a new threshold. If the user wishes to set a new threshold, determine the new threshold experimentally by performing 3 experiments each containing 12 RPA reactions without any HIV-1 DNA present. Set the threshold at the average increase in fluorescence intensity from these experiments plus 3 standard deviations. After completing Section 6, the script JoVE_qRPA_validation_and_quantification.m automatically returns the estimated DNA concentration for each qRPA reaction (in log10 copies). If the script determines that no HIV-1 DNA was present in the sample, the estimated concentration is listed as either &#8220;Negative for HIV&#8221; or &#8220;Invalid,&#8221; depending on whether the fluorescent signal for the internal positive control exceeded the threshold (z &#215; &#963;background + &#916;background). If the user is validating the standard curve with known sample concentrations, the script will also return an additional table similar to that of Table 1.
In order to develop a real-time RPA assay that provides accurate quantification, experimental consistency is crucial. For example, use the same primer and probe aliquots for both the standard curve and validation experiments. Also avoid freeze-thaw cycles by storing the primer and probe aliquots at 4 &#176;C between experiments rather than -20 &#176;C. The template aliquots used for the standard curve and validation experiments are stored in the same manner. RPA enzyme pellets and reagents from the same lot are used according to the manufacturer recommendations. Lastly, because RPA lacks true &#8216;cycles&#8217; to precisely control the rate of amplification, standardization of user steps is absolutely imperative. When assembling reactions, the user must always follow the same steps in the same order, spending approximately the same amount of time on each step. Reactions must always be mixed gently with a new pipette tip, and bubbles must always be eliminated. Before amplification, reactions must be held at a consistent temperature, and the thermal cycler or microscope software must always be prepared before loading reactions to avoid any amplification at sub-optimal temperatures that could influence quantification. Any variation in the initial reaction conditions may lead to inconsistency in experimental outcomes.
When using a microscope to collect data, additional variables must be controlled to minimize variation in fluorescence intensity. All reactions must be placed in the same region on the stage warmer, and the microscope must be focused on the same region of the well for every sample. Even if these practices are followed, fluorescence data collected on a microscope may exhibit variability due to local bright spots that naturally form during RPA reactions, bubble formation within the reaction chambers, or photobleaching resulting from repeated exposure to excitation light. The influence of these variables is evident in fluorescence data collected on the microscope (Figures 4A and 4B), which demonstrate baseline variability, peaks, and troughs. These features are absent from the fluorescence data collected on the thermal cycler (Figures 3A and 3B). Ultimately, data collection on the microscope is for proof-of-principle purposes only and the final assay will be implemented on a field-operable fluorescence reader with more precise geometry and software control that minimizes these variables.
Another important aspect of the qRPA assay development process is consistency in data processing. The protocol described in the methods section uses scripts to process raw fluorescence data (stored in a spreadsheet file) collected from a thermal cycler or microscope. All experiments used to build the standard curve must be formatted identically. When using a thermal cycler to collect data, the same plate layout must be used, and data from wells that do not contain RPA reactions must not be exported. When using a microscope to collect data, the format of the data must match the format of the data automatically exported from the thermal cycler. For example, the no-target-control data must be in cells C2:C61, and data for increasing template concentrations must be in cells D2:D61, E2:E61, etc. If there are multiple replicates of each concentration in an experiment, the 2nd replicate dilution series must be ordered left to right from no target control (NTC) to highest concentration and saved in the columns immediately to the right of the 1st replicate dilution series. For example, in the plate layout used in Section 1.2 with 2 replicates for each sample, fluorescence data for the 1st replicate of each sample in the dilution series must be saved in cells C2:H61 and fluorescence data for the 2nd replicate of each sample in the dilution series must be saved in cells I2:N61. For the plate layout used in Section 1.2, this is the default formatting when exporting data from the thermal cycler software to a spreadsheet.
Representative data provided from HIV-1 qRPA experiments demonstrate proof-of-concept support that RPA may be used for quantification of nucleic acid concentration in unknown samples. Clinically useful HIV-1 viral load tests have a clinical range of at least 4 orders of magnitude, a precision of 0.5 log10 copies, and a limit-of-detection of at least 200 copies19,20. The HIV-1 DNA assay described meets these criteria and is most accurate at low concentrations, as shown in Table 1. Therefore, with the inclusion of a reverse transcriptase step, these results suggest that an HIV-1 RT-RPA assay may have the potential to measure HIV-1 viral load in clinical samples. When developing a qRPA assay, adjusting the algorithm parameters may tune the sensitivity and linear dynamic range depending on clinical needs. Figure 6 shows that adjusting z (a parameter that determines the threshold for positive samples) can influence the sensitivity and accuracy at low and high target concentrations. Furthermore, it may be possible to increase the resolution and accuracy of quantification by incubating reactions at a lower temperature or using less magnesium acetate, thereby decreasing the rate of amplification.
This proof of concept qRPA assay can be used to quantify the concentration of samples containing HIV-1 DNA. The qRPA assay described in this manuscript includes detailed instructions on how to assemble real-time RPA reactions, develop and screen an IPC, and process raw fluorescence data to build a standard curve that can be used to quantify unknown samples. With the detailed instructions included, this protocol may be adapted to quantify DNA concentration in a wide variety of samples.

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><tbody><tr><td>qRPA Assay</td><td></td><td></td><td></td></tr><tr><td>HIV-1 forward primer</td><td>Integrated DNA Technologies</td><td>custom DNA oligos</td><td>5&amp;rsquo;-TGG CAG TAT TCA TTC ACA ATT TTA AAA GAA AAG G-3&amp;rsquo;&amp;nbsp;</td></tr><tr><td>HIV-1 reverse primer</td><td>Integrated DNA Technologies</td><td>custom DNA oligos</td><td>5&amp;rsquo;-CCC GAA AAT TTT GAA TTT TTG TAA TTT GTT TTT G-3&amp;rsquo;&amp;nbsp;</td></tr><tr><td>HIV-1 probe</td><td>BioSearch Technologies&amp;nbsp;</td><td>custom DNA oligos</td><td>5&amp;rsquo;- TGC TAT TAT GTC TAC TAT TCT TTC CCC [SIMA/HEX] GC [THF] C [dT-BHQ1] GTA CCC CCC AAT CCC C -3&amp;rsquo;&amp;nbsp;</td></tr><tr><td>IPC probe</td><td>BioSearch Technologies&amp;nbsp;</td><td>custom DNA oligos</td><td>5&amp;rsquo;-AGG TAG TGA CAA GAA ATA ACA ATA CAG GAC [FAM] T [THF] T [dT-BHQ1] GGT TTT GTA ATT GGA A -3&amp;rsquo;</td></tr><tr><td>RPA exo reagents (pellets, rehydration buffer, magnesium acetate</td><td>TwistDx</td><td>TwistAmp exo</td><td></td></tr><tr><td>PCR tube strips</td><td>BioRad</td><td>TLS0801</td><td></td></tr><tr><td>PCR flat cap tube strips</td><td>BioRad</td><td>TCS0803</td><td></td></tr><tr><td>Micro-seal adhesive</td><td>BioRad</td><td>558/MJ&amp;nbsp;</td><td></td></tr><tr><td>HIV-1 target (pHIV-IRES- eYFP&amp;Delta;Env&amp;Delta;Vif&amp;Delta;Vpr)</td><td></td><td></td><td>custom plasmid, see: Segall, H. I., Yoo, E. &amp;amp; Sutton, R. E. Characterization and detection of artificial replication-competent lentivirus of altered host range. Molecular Therapy 8, 118&amp;ndash;129, doi:10.1016/S1525-0016(03)00134-5 (2003).</td></tr><tr><td>Human male genomic DNA</td><td>Applied Biosystems</td><td>360486</td><td></td></tr><tr><td>96 well cold-block</td><td>Cole Parmer</td><td>EW-36700-48</td><td></td></tr><tr><td>Thermal cycler</td><td>BioRad</td><td>CFX96</td><td></td></tr><tr><td>MiniFuge</td><td>VWR</td><td>93000-196</td><td></td></tr><tr><td>Vortex</td><td>VWR</td><td>58816-121</td><td></td></tr><tr><td>Tris buffer 1.0 M, pH 8.0</td><td>EMD Millipore</td><td>648314</td><td></td></tr><tr><td>EDTA 0.5 M, pH 8.0</td><td>Promega</td><td>V4321</td><td></td></tr><tr><td>Nuclease free water</td><td>Ambion</td><td>AM9937</td><td></td></tr><tr><td>IPC Development</td><td></td><td></td><td></td></tr><tr><td>Cryptosporidium parvum IPC template</td><td>Waterborne Inc</td><td>P102C</td><td>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><td>PCR long forward primer</td><td>Integrated DNA Technologies</td><td>custom DNA oligos</td><td>5&amp;rsquo;-TGG CAG TAT TCA TTC ACA ATT TTA AAA GAA AAG G/ ATC TAA GGA AGG CAG CAG GC-3&amp;rsquo;</td></tr><tr><td>PCR long reverse primer</td><td>Integrated DNA Technologies</td><td>custom DNA oligos</td><td>5&amp;rsquo;- CCC GAA AAT TTT GAA TTT TTG TAA TTT GTT TTT G/ TGC TGG AGT ATT CAA GGC ATA -3&amp;rsquo;</td></tr><tr><td>Phusion High-Fidelty DNA Polymerase</td><td>New England Biolabs</td><td>M0530S</td><td></td></tr><tr><td>Qiaquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>TAE 10X buffer</td><td>EMD Millipore</td><td>574797</td><td></td></tr><tr><td>Agarose</td><td>Sigma Aldrich</td><td>A9539</td><td></td></tr><tr><td>Microscope Experiments</td><td></td><td></td><td></td></tr><tr><td>Upright fluorescence microscope</td><td>Zeiss</td><td>Zeiss Imager.J1</td><td></td></tr><tr><td>Stage heater</td><td>Bioscience Tools</td><td>TC-GSH</td><td></td></tr><tr><td>1-Channel Precision High Stability Temperature Controller</td><td>Bioscience Tools</td><td>TC-1100S</td><td></td></tr><tr><td>FAM/GFP filter cube</td><td>Zeiss</td><td>filter set 38 (000000-1031-346)</td><td>excitation BP 470/40 nm, emission BP 520/50 nm</td></tr><tr><td>HEX filter cube</td><td>Chroma</td><td>49014</td><td>excitation BP 530/30 nm, emission BP 575/40 nm</td></tr><tr><td>Laser cutter</td><td>Engraver&#039;s Network</td><td>VLS3.60</td><td></td></tr><tr><td>1/8&quot; black acrylic</td><td>McMaster Carr</td><td>8505K113</td><td></td></tr><tr><td>1.5 mm clear acrylic</td><td>McMaster Carr</td><td>PD-72268940&amp;nbsp;</td><td></td></tr><tr><td>Super glue</td><td>Office Depot</td><td>Duro super glue&amp;nbsp;</td><td></td></tr><tr><td>PCR grade mineral oil</td><td>Sigma Aldrich</td><td>M8662-5VL</td><td></td></tr><tr><td>Data Analysis</td><td></td><td></td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td></td><td></td></tr><tr><td>MATLAB</td><td>MATLAB</td><td></td><td></td></tr><tr><td>MATLAB script: &quot;JoVE_qRPA_standard_curve.m&amp;rdquo;</td><td>Included in SI</td><td></td><td></td></tr><tr><td>MATLAB script: &quot;JoVE_qRPA_validation_and_quantification.m&amp;rdquo;</td><td>Included in SI</td><td></td><td></td></tr><tr><td>MATLAB script: &quot;JoVE_real_time_intensity_to_excel.m&amp;rdquo;</td><td>Included in SI</td><td></td><td></td></tr><tr><td>Adobe Illustrator</td><td>Adobe</td><td></td><td></td></tr><tr><td>JoVE_qRPA_well.ai</td><td>Included in SI</td><td></td><td></td></tr><tr><td>JoVE_qRPA_base.ai</td><td>Included in SI</td><td></td><td></td></tr><tr><td>AxioVision software</td><td>Zeiss</td><td></td><td></td></tr><tr><td>JoVE_AxioVision_Script.ziscript</td><td>Included in SI</td><td></td><td></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 electrophoresis. In qRPA reactions, the shorter IPC candidate exhibited little amplification (Figure 2B), while the longer candidate consistently amplified when a total of 104 and 105 copies were present (Figure 2C). Thus, the longer candidate was chosen to be the IPC for HIV-1 qRPA experiments.
Real-time qRPA may be performed using the target of interest alone or using both the target and the IPC. Figure 3 shows an experiment on the thermal cycler using both HIV-1 DNA and the C. parvum IPC. In this experiment, 2.6 x 104 copies of IPC DNA were added to each reaction, a concentration close to the IPC limit of detection, to avoid affecting the limit of detection of HIV-1 target DNA. As demonstrated in Figure 3A, which displays HEX fluorescence data corresponding to real-time HIV-1 DNA generation, the time at which detectable amplification begins is inversely proportional to the initial target concentration. Thus, amplification is apparent earlier for high concentrations of HIV-1 DNA and later for low concentrations of HIV-1 DNA. In contrast, detectable amplification of IPC DNA begins at approximately the same time because the starting IPC concentration is the same in all samples, as shown in Figure 3B, which displays FAM fluorescence data corresponding to IPC DNA generation during the same experiment. The rate of fluorescence generation from the IPC is inversely proportional to the concentration of HIV-1 DNA due to competition during amplification.
With the goal of developing a field-operable fluorescence reader with qRPA reactions, qRPA experiments may be performed on an upright fluorescence microscope with a stage heater and 1-Channel Precision High Stability temperature controller. Figure 4A and 4B display HEX and FAM fluorescence data collected on a microscope. Data collected on the microscope using laser-cut chips demonstrate slight variability in baseline fluorescence and crests and troughs that may be due to photobleaching. However, the script determines the cycle threshold using the rate of change of fluorescence during the initial amplification period, which is unaffected by baseline fluorescence or variability in fluorescence after the initial amplification has occurred. As seen in Figure 4C, the standard curve built from this experiment has an r2 coefficient of 0.990. Notably, the IPC is amplified only for low concentrations of HIV-1 DNA. Although this behavior differs from experiments performed on the thermal cycler, all samples are still classified as valid using this method.
Data from multiple experiments using known target concentrations may be compiled to construct a standard curve, which may then be used to assess assay performance or quantify samples with unknown concentrations. Figure 5 shows the data from five experiments and an exponential standard curve generated using the script &#8220;JoVE_qRPA_standard_curve.m&#8221;, relating the initial HIV-1 target concentration to the time at which detectable amplification began. Figure 5 used 5 separate experiments, each containing 2 qRPA reactions at each template concentration to build a standard curve. Note that the exponential fit has a high r2 coefficient of 0.959. The standard curve was then used to predict the concentrations of additional samples with known concentrations for assay validation using the script &#8220;JoVE_qRPA_validation_and_quantification.m&#8221; script. Table 1 shows the quantification results for 5 additional experiments using the standard curve in Figure 5. Note that the algorithm correctly classified all samples containing HIV-1 DNA as positive and 9 out of 10 of the no-target-control samples as negative. In addition, the average predicted concentration was within one order of magnitude of the correct concentration and the standard deviation for predicted concentrations was less than 0.5 log10 (copies per reaction).
<img alt="Figure 1" src="/files/ftp_upload/52620/52620fig1highres.jpg" /> 
Figure 1: Block diagram of quantitative RPA DNA targets. The qRPA assay amplifies two DNA sequences flanked by identical sequences to which primers bind (&#8220;RPA forward&#8221; and &#8220;RPA reverse&#8221;). The amplified sequences are 1) a sequence within the HIV-1 genome (&#8220;HIV-1&#8221;) that is detected with the &#8220;HIV-1 probe&#8221; and 2) an internal positive control sequence (&#8220;IPC (C. parvum)&#8221;) included in each reaction that is detected with the &#8220;IPC probe.&#8221; The IPC was generated via PCR using the Cryptosporidium parvum genome as a template and primers (&#8220;PCR forward&#8221; and &#8220;PCR reverse&#8221;) that contain a region complementary to C. parvum (purple) and a region complementary to HIV-1 (blue). Figure originally published in Analytical Chemistry 2014, reprinted here with permission9.
<img alt="Figure 2" src="/files/ftp_upload/52620/52620fig2.jpg" /> 
Figure 2: Screening of IPC candidates. (A) Two IPC candidates (&#8216;Forward Short&#8217; and &#8216;Forward Long&#8217;) and a known PCR positive control sequence were generated using PCR and visualized on an agarose gel, where 415 bp and 435 bp bands were visible. (B) The short IPC candidate exhibited little amplification during real-time RPA, while (C) the longer IPC candidate amplified consistently when a total of 104 and 105 copies were present. Figure originally published in Analytical Chemistry 2014, reprinted here with permission9. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52620/52620fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52620/52620fig3highres.jpg" /> 
Figure 3: Typical raw fluorescence data generated during co-amplification of HIV-1 DNA and IPC on a thermal cycler. (A) For HIV-1, the onset of detectable amplification, shown by an apparent increase in HEX fluorescence, occurs earlier for high concentrations of HIV-1 DNA. (B) For the IPC, the onset of detectable amplification, shown by an apparent increase in FAM fluorescence, occurs at approximately the same time regardless of the HIV-1 DNA concentration. However, the rate of FAM fluorescence generation is inversely proportional to the amount of HIV-1 DNA present due to competition. Figure originally published in Analytical Chemistry 2014, reprinted here with permission9.
<img alt="Figure 4" src="/files/ftp_upload/52620/52620fig4highres.jpg" /> 
Figure 4: Typical raw fluorescence data generated during co-amplification of HIV-1 DNA and IPC on a microscope. (A) Similar to data generated on the thermal cycler, the onset of detectable HIV-1 amplification, shown by an apparent increase in HEX fluorescence, occurs earlier for high concentrations of HIV-1 DNA. (B) IPC amplification on the microscope, shown by FAM fluorescence, is apparent for low HIV-1 DNA concentrations but not always apparent in samples with high HIV-1 DNA concentrations. (C) A standard curve can be built using the JoVE_standard_curve.m scripts that yields a high r2 coefficient.
<img alt="Figure 5" src="/files/ftp_upload/52620/52620fig5highres.jpg" /> 
Figure 5: Typical standard curve for HIV-1. Using the JoVE_standard_curve.m script, raw HEX fluorescence data generated during HIV-1 amplification is processed to build a standard curve, which may be used to predict the HIV-1 DNA concentration of unknown samples. This standard curve (z = 1) was generated using data from 5 experiments, in which 6 concentrations were tested using 2 replicates at each concentration. All concentrations are given in log10 copies. Figure originally published in Analytical Chemistry 2014, reprinted here with permission9.
<img alt="Figure 6" src="/files/ftp_upload/52620/52620fig6highres.jpg" /> 
Figure 6: Algorithm tunability. Adjusting the algorithm parameters changes the standard curve used to predict the concentration of unknown samples. The JoVE_validation_and_quantification.m script was used to predict the concentration of unknown samples using z = 1 (red) and z = 5 (blue). All concentrations are given in log10 copies. Predictions are more accurate for low DNA concentrations when z = 1; predictions are more accurate for high DNA concentrations when z = 5. By adjusting the algorithm parameters, the qRPA standard curve may be tuned according to clinical needs. Figure originally published in Analytical Chemistry 2014, reprinted here with permission9.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sample Concentration</td>
			<td>Average Predicted Concentration</td>
			<td>Standard Deviation</td>
			<td>Percent of Samples Identified as Positive</td>
		</tr>
		<tr>
			<td>No target controls</td>
			<td>0.1</td>
			<td>0.3</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>1</td>
			<td>0.9</td>
			<td>0.1</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>2</td>
			<td>2.2</td>
			<td>0.5</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>3</td>
			<td>3</td>
			<td>0.1</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>4</td>
			<td>3.7</td>
			<td>0.1</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>5</td>
			<td>4.2</td>
			<td>0.2</td>
			<td>100%</td>
		</tr>
	</tbody>
</table>
Table 1: HIV-1 qRPA assay validation. Using the standard curve in Figure 4, the JoVE_validation_and_quantification.m script was used to predict the concentrations (in log10 copies) of samples for 5 additional experiments. Table originally published in Analytical Chemistry 2014, reprinted here with permission9.

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 ...

development-quantitative-recombinase-polymerase-amplification-assay

Nanyang Technological University

Research

JoVE Journal

Biology

13.6K Views.  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.

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

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

Genetics

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Immunology and Infection

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The use of environmental DNA (eDNA) samples for detecting macrobiota is one such group of methods that is rapidly becoming popular and being implemented in national management programs. Here we focus on the development of species-specific targeted assays for probe-based quantitative PCR (qPCR) applications. Using probe-based qPCR offers greater specificity than is possible with primers alone. Furthermore, the ability to quantify the amount of DNA in a sample can be useful in our understanding of the ecology of eDNA and the interpretation of eDNA detection patterns in the field. Careful consideration is needed in the development and testing of these assays to ensure the sensitivity and specificity of detecting the target species from an environmental sample. In this protocol we will delineate the steps needed to design and test probe-based assays for the detection of a target species; including creation of sequence databases, assay design, assay selection and optimization, testing assay performance, and field validation. Following these steps will help achieve an efficient, sensitive, and specific assay that can be used with confidence. We demonstrate this process with our assay designed for populations of the mucket (Actinonaias ligamentina), a freshwater mussel species found in the Clinch River, USA.

Environmental DNA assays require rigorous design, testing, optimization and validation before the collection of field data can begin. Here, we present a protocol to take users through each step of designing a species-specific, probe-based qPCR assay for the detection and quantification of a target species DNA from environmental samples.

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The ...

Environment

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. 
Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. 
This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories.

Here, we present a protocol for the development and validation of a quantitative PCR method used for the detection and quantification of EHV-2 DNA in equine respiratory fluids. The EHV-2 qRT-PCR validation protocol involves a three-part procedure: development, characterization of qRT-PCR assay alone, and characterization of the whole analytical method.

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 ...

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). 
The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified
simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific.
Published applications of mPCR/RLB include detection of antibiotic resistance genes1,2, typing of methicillin-resistant Staphylococcus aureus3-5 and Salmonella sp6, molecular serotyping of Streptococcus pneumoniae7,8, Streptococcus agalactiae9 and enteroviruses10,11, identification of Mycobacterium sp12, detection of genital13-15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.
The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.

Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction ...

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

Medicine

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.

Detailed instructions are provided to build an open-source, modular fluorimeter that is compatible with many low-cost heaters to perform real-time, quantitative isothermal nucleic acid amplification.

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with ...

Bioengineering

Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify&#160;unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3&#8217;- and 5&#8217;- sequences adjacent to the integrated lentiviral vector.

Linear-amplification mediated (LAM)-PCR is a method developed to identify the exact positions of integrating viral vectors in the genome. The technique has evolved to be the superior method to study clonal dynamics in gene therapy patients, biosafety of novel vector technologies, T-cell diversity, cancer stem cell models, etc.

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with ...

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

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