Questa ricerca è stata finanziata da una sovvenzione della Bill &amp; Melinda Gates Foundation attraverso il Grand Challenges in Global Health Initiative.

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T.+gondii+RP+Promoters+&amp;+Knockdown+Reveal+Molecular+Pathways+Associated+with+Proliferation+and+Cell-Cycle+Arrest.">T. gondii RP Promoters &amp; Knockdown Reveal Molecular Pathways Associated with Proliferation and Cell-Cycle Arrest.</a> PLoS ONE. 5 (11), 15 (2010).</li><li>Segall, H. I., Yoo, E., Sutton, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+detection+of+artificial+replication-competent+lentivirus+of+altered+host+range.">Characterization and detection of artificial replication-competent lentivirus of altered host range.</a> Molecular Therapy. 8 (1), 118-129 (2003).</li><li>Crannell, Z. A., Rohrman, B. 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The authors declare they have no competing financial interests.

Al fine di ottenere dati di quantificazione significativi utilizzando l&#39;algoritmo di MATLAB, l&#39;utente deve selezionare i valori di input appropriati quando richiesto. Dopo l&#39;avvio ogni script ai punti 5 e 6, tutte le variabili di input sono sollecitati automaticamente nella finestra di comando e le uscite sono generati automaticamente. Nella Sezione 5.7 all&#39;utente viene richiesto di selezionare una soglia pista. Il valore della soglia pista colpisce il quadrato del coefficiente di correlazione (r 2)...

Quantitative amplificazione degli acidi nucleici è una tecnica importante per il rilevamento di ambientale, di origine alimentare, e gli agenti patogeni di origine idrica, nonché per la diagnostica clinica. Real time PCR quantitativa (qPCR) è il metodo gold standard per il rilevamento sensibile, specifico e quantitativa degli acidi nucleici, ad esempio, per l&#39;HIV-1 test di carica virale, il rilevamento di batteri patogeni, e lo screening per molti altri organismi 1 - 3. Durante tempo reale qPCR, primer amplificano patogeno DNA in cicli, e un segnale fluorescente viene generato che è proporzionale alla quantità di DNA amplificato nel campione ad ogni ciclo. Un campione contenente una concentrazione sconosciuta di DNA patogeno può essere quantificato utilizzando una curva standard che riguarda la concentrazione di DNA iniziale di campioni standard e il momento in cui il segnale fluorescente raggiunge una certa soglia (cioè, la soglia ciclo o C T). <p class=&quot;Jove_content&quot;&gt; Perché in tempo reale qPCR richiede costose attrezzature cicli termici e diverse ore per ricevere i risultati, tecniche di amplificazione isotermica alternativi, come recombinase polimerasi amplificazione (RPA), sono stati sviluppati 4. Queste piattaforme genere forniscono risultati più rapidi e amplificare acidi nucleici a singolo temperatura inferiore, che può essere realizzato con meno costoso, attrezzature semplici. RPA, che è particolarmente interessante per applicazioni point-of-care, amplifica il DNA in minuti, richiede una temperatura bassa di amplificazione (37 ° C), e rimane attiva in presenza di impurità 5,6. Saggi RPA sono stati sviluppati per una vasta gamma di applicazioni, tra cui l&#39;analisi degli alimenti, degli agenti patogeni, lo screening di farmaci antitumorali, e la rilevazione di agenti biothreat 7-12. Tuttavia, l&#39;uso di RPA per la quantificazione degli acidi nucleici è stata limitata 13,14. In lavori precedenti, è stato shown che in tempo reale RPA quantitativa (qRPA) è fattibile 15. Qui, un protocollo più dettagliata per l&#39;utilizzo in tempo reale quantitativa RPA quantificare campioni sconosciuti utilizzando una curva standard, un metodo che è analogo a quantificazione utilizzando qPCR. Questo protocollo descrive come eseguire una reazione RPA su un termociclatore per rilevare HIV-1 DNA come un proof-of-concept, e come sviluppare un controllo positivo interno (IPC) per garantire che il sistema funzioni correttamente. La raccolta dei dati utilizzando un termociclatore o microscopio e analisi dei dati per la costruzione di una curva standard utilizzando i dati di formazione è anche dettagliato. Infine, il metodo per quantificare i campioni sconosciuti utilizzando la curva standard con uno script personalizzato è dimostrata. Questa tecnica consente qRPA quantificazione dei campioni con concentrazioni sconosciute e ha molti vantaggi rispetto tradizionale in tempo reale 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.

Prima di selezionare una sequenza di servire come IPC negli esperimenti qRPA con l&#39;obiettivo (HIV-1) DNA, controllo interno positivo (IPC) i candidati sono generati e screening per la loro capacità di amplificare in reazioni qRPA senza HIV-1 DNA presente. Candidati IPC sono più lunghi rispetto al target (HIV-1) DNA per prevenire la formazione IPC da fuori competizione HIV-1 formazione amplicon. Come mostrato in Figura 2A, la generazione di due C. candidati parvum IPC sono stati v...

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.

Sviluppo di un Quantitative ricombinasi Polymerase Amplificazione Assay con un controllo positivo interno

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 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.