The authors would like to express their gratitude to Aline Burda Farias for the technical assistance with the vacuum oven, as well as to the administration at the Instituto de Biologia Molecular do Parana (IBMP, Curitiba, Brazil) for allowing access to the said equipment. This work was partially funded by grant CNPq 445954/2020-5.

1. Preparation of stock solutions and gelification mixture
NOTE: Four stock solutions will be prepared (400 mg/mL of melezitose, 400 mg/mL of trehalose, 0.75&#160;mg/mL of lysine, and 200 mg/mL of glycogen) and mixed according to the proportion shown in Table 1 to produce the gelification mixture. Although the protocol describes 10 mL of stock solutions production, it can be adapted for lower or higher volumes.
<ol>
	<li>Melezitose solution
	<ol>
		<li>Weigh...

<ol>
	<li>Lewinsohn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carlos+Chagas+(1879-1934):+the+discovery+of+Trypanosoma+cruzi+and+of+American+trypanosomiasis+(foot-notes+to+the+history+of+Chagas's+disease).">Carlos Chagas (1879-1934): the discovery of Trypanosoma cruzi and of American trypanosomiasis (foot-notes to the history of Chagas's disease).</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 73 (5), 513-523 (1979).</li><li>Bern, 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=Evaluation+and+treatment+of+Chagas+disease+in+the+United+States:+A+systematic+review.">Evaluation and treatment of Chagas disease in the United States: A systematic review.</a> The Journal of American Medical Association. 298 (18), 2171-2181 (2007).</li><li>Pereira, K. S., 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=Chagas'+disease+as+a+foodborne+illness.">Chagas' disease as a foodborne illness.</a> Journal of Food Protection. 72 (2), 441-446 (2009).</li><li>Tanowitz, H. B., 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=Chagas'+disease.">Chagas' disease.</a> Clinical Microbiology Reviews. 5 (4), 400-419 (1992).</li><li>Rassi, A., Marin-Neto, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chagas+disease.">Chagas disease.</a> Lancet. 375 (9723), 1388-1402 (2010).</li><li>Ram&#237;rez, J. 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=Analytical+validation+of+quantitative+real-time+PCR+methods+for+quantification+of+trypanosoma+cruzi+DNA+in+blood+samples+from+chagas+disease+patients.">Analytical validation of quantitative real-time PCR methods for quantification of trypanosoma cruzi DNA in blood samples from chagas disease patients.</a> The Journal of Molecular Diagnostics. 17 (5), 605-615 (2015).</li><li>Rivero, R., 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+detection+of+Trypanosoma+cruzi+by+colorimetric+loop-mediated+isothermal+amplification+(LAMP):+A+potential+novel+tool+for+the+detection+of+congenital+Chagas+infection.">Rapid detection of Trypanosoma cruzi by colorimetric loop-mediated isothermal amplification (LAMP): A potential novel tool for the detection of congenital Chagas infection.</a> Diagnostic Microbiology and Infectious Disease. 89 (1), 26-28 (2017).</li><li>Schijman, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+diagnosis+of+Trypanosoma+cruzi.">Molecular diagnosis of Trypanosoma cruzi.</a> Acta Tropica. 184, 59-66 (2018).</li><li>Besuschio, S. 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=Trypanosoma+cruzi+loop-mediated+isothermal+amplification+(Trypanosoma+cruzi+Loopamp)+kit+for+detection+of+congenital,+acute+and+Chagas+disease+reactivation.">Trypanosoma cruzi loop-mediated isothermal amplification (Trypanosoma cruzi Loopamp) kit for detection of congenital, acute and Chagas disease reactivation.</a> Plos Neglected Tropical Diseases. 14 (8), 0008402 (2020).</li><li>Duffy, T., 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=Accurate+real-time+PCR+strategy+for+monitoring+bloodstream+parasitic+loads+in+chagas+disease+patients.">Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients.</a> Plos Neglected Tropical Diseases. 3 (4), (2009).</li><li>Melo, M. F., 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=Usefulness+of+real+time+PCR+to+quantify+parasite+load+in+serum+samples+from+chronic+Chagas+disease+patients.">Usefulness of real time PCR to quantify parasite load in serum samples from chronic Chagas disease patients.</a> Parasites &amp; Vectors. 8 (1), 154 (2015).</li><li>Parrado, R., 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=Usefulness+of+serial+blood+sampling+and+PCR+replicates+for+treatment+monitoring+of+patients+with+chronic+Chagas+disease.">Usefulness of serial blood sampling and PCR replicates for treatment monitoring of patients with chronic Chagas disease.</a> Antimicrobial Agents and Chemotherapy. 63 (2), 01191 (2019).</li><li>de Rampazzo, R. C. P., 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+ready-to-use+duplex+qPCR+to+detect+Leishmania+infantum+DNA+in+naturally+infected+dogs.">A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs.</a> Veterinary Parasitology. 246, 100-107 (2017).</li><li>Rampazzo, R. C. P., 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=Proof+of+concept+for+a+portable+platform+for+molecular+diagnosis+of+tropical+diseases.">Proof of concept for a portable platform for molecular diagnosis of tropical diseases.</a> The Journal of Molecular Diagnostics. 21 (5), 839-851 (2019).</li><li>Costa, A. D. T., 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=Ready-to-use+qPCR+for+detection+of+Cyclospora+cayetanensis+or+Trypanosoma+cruzi+in+food+matrices.">Ready-to-use qPCR for detection of Cyclospora cayetanensis or Trypanosoma cruzi in food matrices.</a> Food and Waterborne Parasitology. 22, 00111 (2021).</li><li>Sun, Y., 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=Pre-storage+of+gelified+reagents+in+a+lab-on-a-foil+system+for+rapid+nucleic+acid+analysis.">Pre-storage of gelified reagents in a lab-on-a-foil system for rapid nucleic acid analysis.</a> Lab on a Chip. 13, 1509-1514 (2013).</li><li>Kamau, E., 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=Sample-ready+multiplex+qPCR+assay+for+detection+of+malaria.">Sample-ready multiplex qPCR assay for detection of malaria.</a> Malaria Journal. 13, 158 (2014).</li><li>Kasper, J. C., Winter, G., Friess, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+and+further+challenges+in+lyophilization.">Recent advances and further challenges in lyophilization.</a> European Journal of Pharmaceutics and Biopharmaceutics. 85 (2), 162-169 (2013).</li><li>Rosado, P. M. F. S., L&#243;pez, G. L., Seiz, A. M., Alberdi, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+preparing+stabilised+reaction+mixtures,+which+are+totally+or+partially+dried,+comprising+at+least+one+enzyme,+reaction+mixtures+and+kits+containing+said+mixtures.">Method for preparing stabilised reaction mixtures, which are totally or partially dried, comprising at least one enzyme, reaction mixtures and kits containing said mixtures.</a> PubChem. , (2002).</li><li>Ali, N., Bello, G. L., Rossetti, M. L. R., Krieger, M. A., Costa, A. D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+a+fast+and+easy+sample-to-answer+protocol+for+tuberculosis+screening+in+point-of-care+settings:+A+proof+of+concept+study.">Demonstration of a fast and easy sample-to-answer protocol for tuberculosis screening in point-of-care settings: A proof of concept study.</a> PLoS One. 15 (12), 0242408 (2020).</li><li>Murphy, H. R., Lee, S., da Silva, 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=Evaluation+of+an+improved+U.S.+food+and+drug+administration+method+for+the+detection+of+Cyclospora+cayetanensis+in+produce+using+real-time+PCR.">Evaluation of an improved U.S. food and drug administration method for the detection of Cyclospora cayetanensis in produce using real-time PCR.</a> Journal of Food Protection. 80 (7), 1133-1144 (2017).</li><li>Iglesias, N., 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=Performance+of+a+new+gelled+nested+PCR+test+for+the+diagnosis+of+imported+malaria:+comparison+with+microscopy,+rapid+diagnostic+test,+and+real-time+PCR.">Performance of a new gelled nested PCR test for the diagnosis of imported malaria: comparison with microscopy, rapid diagnostic test, and real-time PCR.</a> Parasitology Research. 113 (7), 2587-2591 (2014).</li><li>Alonso-Padilla, J., Gallego, M., Schijman, A. G., Gascon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+diagnostics+for+Chagas+disease:+up+to+date+and+novel+methodologies.">Molecular diagnostics for Chagas disease: up to date and novel methodologies.</a> Expert Review of Molecular Diagnostics. 17 (7), 699-710 (2017).</li></ol>

Recent years have highlighted the need to find more sensitive and specific technologies to help diagnose tropical and neglected diseases. Although important for epidemiological control, parasitological (optical microscopy) and serological tests have limitations, especially regarding sensitivity and point-of-care applicability. DNA amplification techniques such as PCR, isothermal amplification, and respective variations have long been used in laboratory settings, but technological hurdles preclude it from being used in fi...

Chagas disease was discovered in the early 20th century in rural regions of Brazil, where poverty was widespread1,2. Even today, the disease continues to be connected to social and economic determinants of health in the Americas. Chagas disease is biphasic, comprising an acute and a chronic phase. It is caused by infection by the Trypanosoma cruzi parasite, being transmitted by insect vectors, blood transfusions via congenital route, or oral ingestion of contaminated food3,4.
The diagnostic of Chagas disease can be made through the observation of clinical symptoms (especially the Roma&#241;a sign), blood smear microscopy, serology, and molecular tests such as real-time PCR (qPCR) or isothermal amplification4,5,6,7,8,9. Clinical symptoms and blood smear microscopy are used in suspected cases of acute infections, while the search for antibodies is used as a screening tool in asymptomatic patients. Because of its sensitivity and specificity, qPCR has been suggested to be used as a monitoring tool for chronic patients, for acute patients undergoing treatment measuring the parasite load in the blood, and as a surrogate marker of therapeutic failure6,8,10,11,12. Although more sensitive and specific than currently available tests, qPCR is effectively prevented from being known as diagnostic tools in underprivileged regions worldwide due to the requirement of freezing temperatures for transportation and storage13,14,15.
To circumvent this obstacle, conservation techniques such as lyophilization and gelification have been explored16,17. While lyophilization provides conservation for years, it requires specially made reagents without the presence of glycerol, which is commonly used for enzyme stabilization/conservation18. While gelification has been shown to provide conservation for months, it allows the use of regular reagents19. The gelification solution comprises four components, each with specific roles in the process: the sugars trehalose and melezitose protect the biomolecules during the desiccation process by reducing free water molecules in the solution, glycogen produces a broader protective matrix, and the amino acid lysine is used as a free radical scavenger to inhibit the oxidizing reactions between the biomolecule&#39;s carboxyl, amino and phosphate groups. These components define a sol-gel mixture that prevents the loss of the tertiary or quaternary structure during the desiccation process, thus helping to maintain the biomolecules&#39; activity upon rehydration19. Once stabilized inside the reaction tubes, the reactions can be stored for a few months at 2-8 &#176;C or a few weeks at 21-23 &#176;C instead of the regular -20 &#176;C. This approach has already been incorporated in tests designed to help diagnose diseases such as Chagas disease, malaria, leishmaniasis, tuberculosis, and cyclosporiasis13,14,15,20.
The present work describes all the steps to prepare the required solutions for the gelification procedure, the pitfalls in the process, and the expected final aspect of a ready-to-use gelified qPCR inside eight-tube strips. The same protocol can be adapted for single tubes or 96-well plates. Finally, the detection of T. cruzi DNA will be shown as a control run.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bentonite clay bags (activated)</td>
			<td>Embamat Global Packaging Solutions (Barcelona, Spain)</td>
			<td>026157/STD</td>
			<td>Not to be confused with silica gel packs</td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Amersham Bioscience</td>
			<td>Cat# US16445</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysine</td>
			<td>Acros Organic</td>
			<td>Cat# 365650250</td>
			<td></td>
		</tr>
		<tr>
			<td>Melezitoze</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# 63620</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>preferred vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oligonucleotides</td>
			<td>preferred vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR mastermix</td>
			<td>preferred vendor or Instituto de Biologia Molecular do Paran&#225; (IBMP, Curitiba, Brazil)</td>
			<td>Chagas NAT kit</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR thermocycler</td>
			<td>preferred vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>software for vacuum oven</td>
			<td>Memmert Gmbh</td>
			<td>Celsius v10.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Trehalose</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# T9531</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypanosoma cruzi DNA</td>
			<td>from in-house cultivated parasites, or purchased from accredited vendors such as ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum oven</td>
			<td>Memmert Gmbh</td>
			<td>VO-400</td>
			<td></td>
		</tr>
	</tbody>
</table>

ready-to-use qpcr for detection of dna from trypanosoma cruzi or other pathogenic organisms

Real-time PCR (qPCR) is a remarkably sensitive and precise technique&#160;that allows for amplifying&#160;minute amounts of nucleic acid targets from a multitude of samples. It has been extensively used in many research areas and achieved industrial application in fields such as human diagnostics and trait selection in crops of genetically modified organisms (GMO) crops. However, qPCR is not an error-proof technique. Mixing all reagents into a single master mix subsequently distributed onto 96 wells of a regular qPCR plate might lead to operator mistakes such as incorrect mixing of reagents or inaccurate dispensing into the wells. Here, a technique called gelification is presented, whereby most of the water present in the master mix is substituted by reagents that form a sol-gel mixture when submitted to a vacuum. As a result, qPCR reagents are effectively preserved for a few weeks at room temperature or a few months at 2-8 oC. Details of preparing each solution are shown here along with the expected aspect of a gelified reaction designed to detect&#160;T. cruzi satellite DNA (satDNA). A similar procedure can be applied to detect other organisms. Starting a gelified qPCR run is as simple as removing the plate from the refrigerator, adding the samples to their respective wells, and starting the run, thus decreasing the setup time of a full-plate reaction to the time it takes to load the samples. Additionally, gelified PCR reactions can be produced and controlled for quality in batches, saving time and avoiding common operator mistakes while running routine PCR reactions.

Three of the reagents that form the gelification mixture are easily solubilized upon vigorous vortexing. However, glycogen requires careful vortexing to ensure the powder has been completely solubilized. Unfortunately, vigorous vortexing produces lots of bubbles, which makes it difficult to determine the actual volume of the solution (Figure 1A-B). Therefore, it is essential to let the glycogen solution rest in the refrigerator until most of the solution tra...

Watch this Scientific Journal Video about Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms at JoVE.com

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

The present work describes the steps for producing ready-to-use qPCR for T. cruzi DNA detection that can be pre-loaded on the reaction vessel and stored in the refrigerator for several months.

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

Real-time PCR (qPCR) is a remarkably sensitive and precise technique&#160;that allows for amplifying&#160;minute amounts of nucleic acid targets from a ...

The gelification protocol produces ready to use qPCR reactions that decrease the time and steps required to start a run. This protocol allows qPCR reactions to be produced and controlled for quality and large quantities at once, decreasing the chances of operator error during recipe calculations or aliquoting into wells. This method can be applied to any qPCR that is already fully optimized in the conventional frozen format.Visual demonstration of this method is important because two of the main quality control steps are visual. To prepare the melezitose solution, weigh four grams of melezitose in a 15 milliliter plastic tube, add six milliliters of nuclease free water, and vortex at maximum speed until the powder is solubilized. Make the final volume to 10 milliliters with nuclease free water, then label and store the solution at two to eight degrees Celsius for up to six months.To prepare the trehalose solution, weigh four grams of trehalose in a 15 milliliter plastic tube, add six milliliters of nuclease free water and vortex at the maximum speed until the powder is solubilized. Then make up the volume to 10 milliliters with nuclease free water and filter the solution through a 0.2 micron filter. Label and store the solution at two to eight degrees Celsius for up to six months.To prepare the glycogen solution, weigh two grams of glycogen in a 15 milliliter tube, add six milliliters of nuclease free water, and vortex until the powder is solubilized. Keep the solution at two to eight degrees Celsius for eight to 12 hours because the solubilization of glycogen produces lots of bubbles. The following day, removing the solution from incubation, make up the volume to 10 milliliters with nuclease free water and store the labeled solution at two to eight degrees Celsius for up to six months.To prepare the lysine solution, weigh 7.5 milligrams of lysine in a 15 milliliter tube, add six milliliters of nuclease free water, and vortex until the powder is solubilized. Make up the volume to 10 milliliters with nuclease free water. Filter the solution through a 0.2 micron filter and store at two to eight degrees Celsius for up to six months.To prepare the gelification mixture, mix the appropriate volumes of stock solutions in a 50 milliliter plastic tube. Mix the reagents by inverting the tube 10 times and store the labeled solution at two to eight degrees Celsius for up to three months. To prepare the qPCR master mix for gelification, thaw the reagents in a refrigerated container.Then mix appropriate volumes of the reagents in a 1.5 milliliter tube to prepare enough master mix for an eight tube strip or a 96 well plate. Next pipette 18.5 microliters of the gelification master mix onto each reaction well and place the tubes or plate in the heat conductive support inside the vacuum oven. If using 96 well plates, place one bentonite clay bag in the oven for every two 96 well plates for water absorption.When the cycle is completed, check the tubes or plates for proper gelification of the reagents by ensuring that the volume is visibly reduced to about five microliters and the liquids do not move upon tapping the tubes or plates with fingers. Seal and store the tubes or plates at two to eight degrees Celsius for eight to 12 hours before use. To use the gelified qPCR, remove the tube strip or plate from the refrigerator and open it in a workstation for sample manipulation.Add 15 microliters of nuclease free water and five microliters of DNA sample to each reaction vessel. Seal the tubes or plates, run the desired experiment, and perform regular data analysis. In the present protocol, the temperature inside the chamber is kept constant at 30 degrees Celsius, while the pressure varies between 910 to 930 millibar and near vacuum.After the vacuum cycle, the master mix inside the tube decreases in volume, becomes gelified at the bottom and does not splatter on the walls when the tubes are tapped. If the gelification is incomplete, the liquid spatters on the tube walls when the tubes are tapped. Trypanosoma cruzi-DNA detection using published oligonucleotide sequences is shown here.Detection of the same sample when gelification was not correctly executed, resulted in a loss of sensitivity. The most critical step is the calculation of reagents volume prior to gelification. The operator must remember to not add water, since it will be removed during the process.If the protocol steps are followed closely, operators with basic laboratory and molecular biology skills will have no difficulty performing the gelification process.

ready-to-use-qpcr-for-detection-dna-from-trypanosoma-cruzi-or-other

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Biochemistry

1.4K vistas.  Fundação Oswaldo Cruz (Fiocruz). El protocolo de gelificación produce reacciones de qPCR listas para usar que disminuyen el tiempo y los pasos necesarios para iniciar una carrera. Este protocolo permite que las reacciones de qPCR se produzcan y controlen para determinar la calidad y grandes cantidades a la vez, disminuyendo las posibilidades de error del operador durante los cálculos de recetas o la alícuota en pozos. Este método se puede aplicar a cualquier qPCR que ya esté totalmente optimizada en el formato congelado...