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 4 g of melezitose in a 15 mL plastic tube, add 6 mL of nuclease-free water, and vortex at the maximum speed of the instrument until the powder is solubilized. 
		&#8203;NOTE: More water can be added to facilitate solubilization, taking care not to exceed the final volume (see below).</li>
		<li>Make up the final volume to 10 mL with nuclease-free water. Label and store at 2-8 &#176;C for up to 6 months.</li>
	</ol>
	</li>
	<li>Trehalose solution
	<ol>
		<li>Weigh 4 g of trehalose in a 15 mL plastic tube, add 6 mL of nuclease-free water, and vortex at the maximum speed of the instrument until the powder is solubilized. 
		NOTE: More water can be added to facilitate solubilization, taking care not to exceed the final volume (see below).</li>
		<li>Make up the volume to 10 mL with nuclease-free water and filter the solution through a 0.2 &#181;m filter. Label and store at 2-8 &#176;C for up to 6 months.</li>
	</ol>
	</li>
	<li>Glycogen solution
	<ol>
		<li>Weigh 2 g of glycogen in a 15 mL plastic tube, add 6 mL of nuclease-free water, and vortex at the maximum speed of the instrument until the powder is solubilized. 
		NOTE: More water can be added to facilitate solubilization, taking care not to exceed the final volume (see below).</li>
		<li>Keep the solution at rest at 2-8 &#176;C for 8-12 h because the solubilization of glycogen produces lots of bubbles (Figure 1). Make up the volume to 10 mL with nuclease-free water. Label and store at 2-8 &#176;C for up to 6 months.</li>
	</ol>
	</li>
	<li>Lysine solution
	<ol>
		<li>Weigh 7.5 mg of lysine in a 15 mL plastic tube, add 6 mL of nuclease-free water. Vortex at the maximum speed of the instrument until the powder is solubilized. 
		NOTE: More water can be added to facilitate solubilization, taking care not to exceed the final volume (see below).</li>
		<li>Make up the volume to 10 mL with nuclease-free water and filter the solution through a 0.2 &#181;m filter. Transfer the solution to an amber flask or protect it from light. Label and store at 2-8 &#176;C degrees for up to 6 months.</li>
	</ol>
	</li>
	<li>Gelification Mixture (GM)
	<ol>
		<li>In a 50 mL plastic tube, mix the volumes of stock solutions according to Table 1.</li>
		<li>Mix the reagents by ten end-to-end inversions of the tube. 
		NOTE: There is no need for a filtration step if this step is performed in a laminar flow safety hood. If this step is not performed in a clean environment, filter the solution through a 0.2 &#181;m filter before transferring it to an amber flask.</li>
		<li>Transfer the solution to an amber flask or protect it from light. Label and store at 2-8 &#176;C for up to 3 months. 
		NOTE: As a quality control step for preparing the gelification mixture, ensure that the measured pH, conductivity, and density values are within the following ranges: pH 5.55-6.66; conductivity 0.630-0.757 mS/cm; and density 1.08-1.11 g/cm3. All measurements should be taken at 25 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Preparation of qPCR master mix for gelification
NOTE: In this step, the qPCR master mix for gelification is prepared. Hence, water is not added to the mix but instead, the gelification mixture is added (Table 2).
<ol>
	<li>Thaw the reagents in a refrigerated container. Mix the reagents in a 1.5 mL tube according to Table 2. An example of a reaction with a final volume of 25 &#181;L containing 5 &#181;L of DNA sample is shown here. 
	NOTE: The DNA sample is not added to the mixture in this step; it is used here solely to calculate the final volumes of each reagent of the qPCR master mix. DNA samples should be added right before starting the run (see step 4 below).</li>
</ol>
3. Gelification of the reagents on the reaction vessels
<ol>
	<li>Appropriately multiply the volumes shown in Table 2 for preparation of an eight-tube strip or a 96-well plate.</li>
	<li>Pipet 18.5 &#181;L of the gelification master mix shown in Table 2 onto each reaction well. 
	NOTE: This volume represents the volumes of oligonucleotide mix, PCR buffer, and gelification mixture used for one reaction (according to Table 2) and will vary depending on the concentration of the reagents and the volume needed for one reaction. The final volume in the gelification master mix is different from the volume in a regular master mix because water is not added.</li>
	<li>Place the tubes/plate in the heat-conductive support (e.g., aluminum) inside the vacuum oven. 
	NOTE: The heat-conductive support is optional. The operator must ensure that the bottom of the tubes is in contact with the vacuum oven shelf to allow fast thermal equilibrium.</li>
	<li>Place one bentonite clay bag for every two 96-well plates. 
	NOTE: Bentonite clay bags are used to absorb the removed water from the gelification master mix by the differential pressure exerted by the vacuum. Bentonite clay bags were found to be unnecessary for less than two 96-well plates.</li>
	<li>Subject the tubes/plate containing the gelification master mix to three vacuum cycles (30 &#177; 5 mBar) of 30 min each, alternating with vacuum release until the atmospheric pressure is achieved (900-930 mBar), under controlled temperature (30 &#176;C &#177; 1 &#176;C) (Figure 2). 
	NOTE: The instrument uses software to control the parameters, and an example of the cycle is shown in Figure 2. The user must create the profile for the run, indicating the chosen parameters.</li>
	<li>When the cycle is completed, check the tubes/plates for proper gelification of the reagents by ensuring that the volume is visibly reduced (Figure 3) and that the liquids do not move upon tapping the tubes/plates with fingers. 
	NOTE: If gelification did not occur, the solution would splatter on the tube walls when tubes are tapped (Figure 3).</li>
	<li>Seal and store the tubes/plates at 2-8 &#176;C for 8-12 h before use.</li>
</ol>
4. Using a gelified qPCR
<ol>
	<li>Remove the tube strip or plate from the refrigerator and open it in a workstation for sample manipulation. Add 15 &#181;L of nuclease-free water to each reaction vessel. 
	NOTE: The volume of gelified reagents is considered to be about 5 &#181;L. So, together with the DNA sample volume (see below), the final reaction volume is 25 &#181;L.</li>
	<li>Add 5 &#181;L of DNA sample. 
	NOTE: Any qPCR-quality DNA template might be used. In the present work, DNA was extracted from 108 T. cruzi epimastigotes (strain Dm28c) and was serially diluted at a 1:10 ratio using TE buffer.</li>
	<li>Seal the tubes/plates and proceed to the equipment of choice. Run the experiment and proceed to regular data analysis.</li>
</ol>

<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. 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The authors declare no conflicts of interest.

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 ...

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

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1.4K Views.  Fundação Oswaldo Cruz (Fiocruz). 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.

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

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions.
We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to produce adenosine triphosphate (ATP). The isolation of the intact F1-ATPase from its native source is an essential prerequisite to characterize the enzyme&#39;s protein composition, kinetic parameters, and sensitivity to inhibitors. A highly pure and homogeneous F1-ATPase can be used for structural studies, which provide insight into molecular mechanisms of ATP synthesis and hydrolysis. This article describes a procedure for the purification of the F1-ATPase from Trypanosoma brucei, the causative agent of African trypanosomiases. The F1-ATPase is isolated from mitochondrial vesicles, which are obtained by hypotonic lysis from in vitro cultured trypanosomes. The vesicles are mechanically fragmented by sonication and the F1-ATPase is released from the inner mitochondrial membrane by the chloroform extraction. The enzymatic complex is further purified by consecutive anion exchange and size-exclusion chromatography. Sensitive mass spectrometry techniques showed that the purified complex is devoid of virtually any protein contaminants and, therefore, represents suitable material for structure determination by X-ray crystallography or cryo-electron microscopy. The isolated F1-ATPase exhibits ATP hydrolytic activity, which can be inhibited fully by sodium azide, a potent inhibitor of F-type ATP synthases. The purified complex remains stable and active for at least three days at room temperature. Precipitation by ammonium sulfate is used for long-term storage. Similar procedures have been used for the purification of F1-ATPases from mammalian and plant tissues, yeasts, or bacteria. Thus, the presented protocol can serve as a guideline for the F1-ATPase isolation from other organisms.

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

This protocol describes the purification of F1-ATPase from the cultured insect stage of Trypanosoma brucei. The procedure yields a highly pure, homogeneous, and active complex suitable for structural and enzymatic studies.

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.

Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing &#946;-galactosidase

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many non-endemic countries due to the increase in migration. This disease affects nearly 8 million people, with new cases estimated at 50,000 per year. In the 1960s and 70s, two drugs for ChD treatment were introduced: nifurtimox and benznidazole (BZN). Both are effective in newborns and during the acute phase of the disease but not in the chronic phase, and their use is associated with important side effects. These facts underscore the urgent need to intensify the search for new drugs against T. cruzi.
T. cruzi is transmitted through hematophagous insect vectors of the Reduviidae and Hemiptera families. Once in the mammalian host, it multiplies intracellularly as the non-flagellated amastigote form and differentiates into the trypomastigote, the bloodstream non-replicative infective form. Inside the insect vector, trypomastigotes transform into the epimastigote stage and multiply through binary fission.
This paper describes an assay based on measuring the activity of the cytoplasmic &#946;-galactosidase released into the culture due to parasites lysis by using the substrate, chlorophenol red &#946;-D-galactopyranoside (CPRG). For this, the T. cruzi Dm28c strain was transfected with a &#946;-galactosidase-overexpressing plasmid and used for in vitro pharmacological screening in epimastigote, trypomastigote, and amastigote stages. This paper also describes how to measure the enzymatic activity in cultured epimastigotes, infected Vero cells with amastigotes, and trypomastigotes released from the cultured cells using the reference drug, benznidazole, as an example. This colorimetric assay is easily performed and can be scaled to a high-throughput format and applied to other T. cruzi strains.

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing &#946;-galactosidase

We describe a high-throughput colorimetric assay measuring &#946;-galactosidase activity in three life cycle stages of Trypanosoma cruzi, the causative agent of Chagas disease. This assay can be used to identify trypanocidal compounds in an easy, fast, and reproducible manner.

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...