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/pubmed?term=((Carlos+Chagas+%281879-1934%29%3A+the+discovery+of+Trypanosoma+cruzi+and+of+American+trypanosomiasis+%28foot-notes+to+the+history+of+Chagas%27s+disease%29%5BTitle%5D)+AND+%22Transactions+of+the+Royal+Society+of+Tropical+Medicine+and+Hygiene%22%5BJournal%5D)">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/pubmed?term=((Evaluation+and+treatment+of+Chagas+disease+in+the+United+States%3A+A+systematic+review%5BTitle%5D)+AND+%22The+Journal+of+American+Medical+Association%22%5BJournal%5D)">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/pubmed?term=((Chagas%27+disease+as+a+foodborne+illness%5BTitle%5D)+AND+%22Journal+of+Food+Protection%22%5BJournal%5D)">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/pubmed?term=((Chagas%27+disease%5BTitle%5D)+AND+%22Clinical+Microbiology+Reviews%22%5BJournal%5D)">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/pubmed?term=((Chagas+disease%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Chagas disease.</a> Lancet. 375 (9723), 1388-1402 (2010).</li>
<li>Ramírez, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analytical+validation+of+quantitative+real-time+PCR+methods+for+quantification+of+trypanosoma+cruzi+DNA+in+blood+samples+from+chagas+disease+patients%5BTitle%5D)+AND+%22The+Journal+of+Molecular+Diagnostics%22%5BJournal%5D)">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/pubmed?term=((Rapid+detection+of+Trypanosoma+cruzi+by+colorimetric+loop-mediated+isothermal+amplification+%28LAMP%29%3A+A+potential+novel+tool+for+the+detection+of+congenital+Chagas+infection%5BTitle%5D)+AND+%22Diagnostic+Microbiology+and+Infectious+Disease%22%5BJournal%5D)">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/pubmed?term=((Molecular+diagnosis+of+Trypanosoma+cruzi%5BTitle%5D)+AND+%22Acta+Tropica%22%5BJournal%5D)">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/pubmed?term=((Trypanosoma+cruzi+loop-mediated+isothermal+amplification+%28Trypanosoma+cruzi+Loopamp%29+kit+for+detection+of+congenital%2C+acute+and+Chagas+disease+reactivation%5BTitle%5D)+AND+%22Plos+Neglected+Tropical+Diseases%22%5BJournal%5D)">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/pubmed?term=((Accurate+real-time+PCR+strategy+for+monitoring+bloodstream+parasitic+loads+in+chagas+disease+patients%5BTitle%5D)+AND+%22Plos+Neglected+Tropical+Diseases%22%5BJournal%5D)">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/pubmed?term=((Usefulness+of+real+time+PCR+to+quantify+parasite+load+in+serum+samples+from+chronic+Chagas+disease+patients%5BTitle%5D)+AND+%22Parasites+%26+Vectors%22%5BJournal%5D)">Usefulness of real time PCR to quantify parasite load in serum samples from chronic Chagas disease patients.</a> Parasites & Vectors. 8 (1), 154(2015).</li>
<li>Parrado, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Usefulness+of+serial+blood+sampling+and+PCR+replicates+for+treatment+monitoring+of+patients+with+chronic+Chagas+disease%5BTitle%5D)+AND+%22Antimicrobial+Agents+and+Chemotherapy%22%5BJournal%5D)">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/pubmed?term=((A+ready-to-use+duplex+qPCR+to+detect+Leishmania+infantum+DNA+in+naturally+infected+dogs%5BTitle%5D)+AND+%22Veterinary+Parasitology%22%5BJournal%5D)">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/pubmed?term=((Proof+of+concept+for+a+portable+platform+for+molecular+diagnosis+of+tropical+diseases%5BTitle%5D)+AND+%22The+Journal+of+Molecular+Diagnostics%22%5BJournal%5D)">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/pubmed?term=((Ready-to-use+qPCR+for+detection+of+Cyclospora+cayetanensis+or+Trypanosoma+cruzi+in+food+matrices%5BTitle%5D)+AND+%22Food+and+Waterborne+Parasitology%22%5BJournal%5D)">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/pubmed?term=((Pre-storage+of+gelified+reagents+in+a+lab-on-a-foil+system+for+rapid+nucleic+acid+analysis%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">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/pubmed?term=((Sample-ready+multiplex+qPCR+assay+for+detection+of+malaria%5BTitle%5D)+AND+%22Malaria+Journal%22%5BJournal%5D)">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/pubmed?term=((Recent+advances+and+further+challenges+in+lyophilization%5BTitle%5D)+AND+%22European+Journal+of+Pharmaceutics+and+Biopharmaceutics%22%5BJournal%5D)">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., López, G. L., Seiz, A. M., Alberdi, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Method+for+preparing+stabilised+reaction+mixtures%2C+which+are+totally+or+partially+dried%2C+comprising+at+least+one+enzyme%2C+reaction+mixtures+and+kits+containing+said+mixtures%5BTitle%5D)+AND+%22PubChem%22%5BJournal%5D)">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/pubmed?term=((Demonstration+of+a+fast+and+easy+sample-to-answer+protocol+for+tuberculosis+screening+in+point-of-care+settings%3A+A+proof+of+concept+study%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Evaluation+of+an+improved+U.S.+food+and+drug+administration+method+for+the+detection+of+Cyclospora+cayetanensis+in+produce+using+real-time+PCR%5BTitle%5D)+AND+%22Journal+of+Food+Protection%22%5BJournal%5D)">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/pubmed?term=((Performance+of+a+new+gelled+nested+PCR+test+for+the+diagnosis+of+imported+malaria%3A+comparison+with+microscopy%2C+rapid+diagnostic+test%2C+and+real-time+PCR%5BTitle%5D)+AND+%22Parasitology+Research%22%5BJournal%5D)">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/pubmed?term=((Molecular+diagnostics+for+Chagas+disease%3A+up+to+date+and+novel+methodologies%5BTitle%5D)+AND+%22Expert+Review+of+Molecular+Diagnostics%22%5BJournal%5D)">Molecular diagnostics for Chagas disease: up to date and novel methodologies.</a> Expert Review of Molecular Diagnostics. 17 (7), 699-710 (2017).</li>
</ol>

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 field settings. One of the main obstacles is the need for temperatures of -20 &#176;C for transportation and storage of the reagents. To remediate this situation, techniques such as lyophilization and gelification have been used to store PCR reactions out of the freezer16,18,19.
The present work shows all the steps necessary to gelify a qPCR reaction to detect T. cruzi DNA inside the reaction vessel, be it tubes, tube strips, microfluidic chips, or plates. Preliminary studies using an RT-LAMP reaction suggest that the gelification technique may also be used to preserve and shield other nucleic acid amplification and modification enzymes, as described by Rosado et al.19. Although relatively straightforward, the two steps that cause most of the operator mistakes in qPCR routines are (a) the preparation of glycogen and melezitose solutions and (b) calculation of the volume of the reaction mix to be added to each reaction tube before the vacuum step. First, the glycogen solution must be refrigerated overnight before the final volume adjustment, and the melezitose solution must be vigorously vortexed (possibly with mild heating at 50 &#176;C) for complete solubilization. Second, the researcher planning the experiment must be aware that the reagents&#39; volumes calculated pre-vacuum might be uneven since water is not added to round up to the reaction volume. The actual reaction volume will be obtained when the gelified reaction is re-solubilized by the addition of sample and water, before running the PCR.
The biggest limitation of the method is the stability of the reactions, which is around 6-8 months at 2-8 &#176;C14,15; it is considerably shorter than lyophilized reactions, which may remain stable for years18. Depending on the specificity of the oligonucleotide sequences, unspecific binding and amplification might occur, which should be carefully examined by the researchers. For example, Costa and collaborators report that the annealing temperature of the gelified qPCR for detection of C. cayetanensis had to be adjusted in +1 &#176;C to avoid unspecific amplifications15,21. Similarly, researchers should avoid using enzymes that might be regulated by or use the gelification components as substrates.
The gelification technique is particularly useful because of its ease of use in the laboratory routine as well as an introduction into a production line16,19,22 allowing smooth quality control; the latter in turn enables robust and comparable data across multiple operators and effectively eliminates common operator mistakes at crucial steps, with a bonus of eliminating freezing temperature requirements during transportation and storage. Preliminary studies suggest that eliminating the cold chain would result in an overall reduction of cost by up to 20% for a qPCR test14. Elimination of the cold chain also makes it feasible to implement qPCR as a confirmatory test for neglected diseases such as Chagas disease in underdeveloped regions, thus favoring their epidemiological control23.
Finally, the gelification protocol streamlines the use of qPCR tests as it only requires the user to add water and the extracted T. cruzi DNA, avoiding errors during reagent handling, and decreasing the set-up time as well as the possibility of the reagent&#39;s contamination. Such characteristics provide efficiency for a routine diagnostic laboratory, speeding the delivery of results to patients and increasing the reliability of the diagnosis. Lastly, because it dispenses the need for a -20 &#176;C cold chain, it is suitable for diagnostic use in low-resource environments.

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><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&amp;aacute; (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 trapped within the bubbles has moved down to the main solution body. Considering the production protocols and the lab routine, gelified plates are kept in the refrigerator overnight (or around 8-12 h), resulting in the settling of most of the bubbles, thus making it easier to determine the correct volume and adjust to the desired final volume (Figure 1C). Note the difference in the volume of bubbles between the glycogen tubes in Figures 1B-C, respectively, right after the solubilization and after overnight settling.
Once the gelification mixture is added to the qPCR master mix in substitution for water (Table 2), the tube strips or plates are ready to go to the vacuum oven. The shelves of the vacuum oven contain a Peltier heating element, ensuring that any tubes that are in contact with it remain at the same temperature. In the present protocol, the temperature inside the chamber is kept constant at 30 &#176;C, while the pressure varies between 910-930 mBar (atmospheric pressure) and 30 mBar (near-vacuum). Figure 2 shows these two variables plotted over time, showing the constant temperature (green line, upper panel) and the variation of pressure (red line, lower panel). After the cycles are finished, the master mix inside the well decreases in volume and becomes gelified at the bottom, i.e., without moving or splashing when the tubes are tapped with fingers (Figure 3). The tubes can now be capped and stored at 2-8 &#176;C. The reactions will fail to gelify if the gelification mixture (Table 1) is incorrectly prepared; the proposed quality control step should see the fault before mixing the gelification mixture with qPCR reagents.
To be used, the gelified reagents inside the tubes/plates must be resuspended in nuclease-free water and the DNA sample diluted usually in water or TE buffer. The resuspension of the reagents of the sol-gel mixture is achieved during the first step of denaturation of the qPCR thermal protocol (usually, 5-10 min at 95 &#176;C), so no extra step is required. Figure 4A shows representative traces of the qPCR detection of T. cruzi DNA using published oligonucleotide sequences15. Suboptimal results include loss of sensitivity, which can be tested with a dilution curve of a solution with a known concentration of genomic targets and loss of specificity, which can be tested with a panel of related trypanosomatid organisms. Figure 4B shows the loss of sensitivity that might arise when the gelification process is not correctly executed or when the reaction loses its stability after being stored at 2-8 &#176;C for more than 6 months.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63316/63316fig01.jpg" /> 
Figure 1: Solubilization of glycogen produces lots of bubbles. Because glycogen produces too many bubbles during solubilization, the glycogen solution must be kept at rest before adjusting to the final volume. (A) Bubbles formed during vortexing. (B) All of the powder was solubilized, but it is not possible to determine the final volume because of the excess bubbles. (C) After 12 h in the refrigerator (tube in the middle), the volume of bubbles is reduced. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63316/63316fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63316/63316fig02.jpg" /> 
Figure 2: Vacuum cycling (lower panel) and temperature control (upper panel). Representative traces of temperature (upper panel) and pressure (lower panel) variation are shown. Black lines represent the programmed variations, whereas the green and red lines represent actual readings of the instrument. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63316/63316fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63316/63316fig03.jpg" /> 
Figure 3: Aspects of gelified master mix inside an eight-tube strip. (A) qPCR master mixes before the vacuum exposure. (B) Liquid splatters on the tube walls because of incomplete gelification (only one vacuum cycle). (C) Gelified qPCR reagents with a clear visible reduction in volume. The liquid does not splatter on the walls when the tubes are tapped. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63316/63316fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63316/63316fig04.jpg" /> 
Figure 4: Representative traces of gelified master mixes detecting DNA from T. cruzi epimastigotes. DNA extracted from T. cruzi epimastigotes (108 cells) was serially diluted at a 1:10 ratio, and the DNA concentrations ranging between 104 and 100 cells were subjected to detection using a gelified qPCR. (A) The expected result of correctly gelified qPCR (B) Detection of the same samples using a plate where the gelification was not properly executed, resulting in loss of sensitivity. Note that the lower concentrations are detected less frequently in panel B. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63316/63316fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Stock concentration</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>Melezitose</td>
			<td>400 mg/mL</td>
			<td>3 mL</td>
		</tr>
		<tr>
			<td>Treahlose</td>
			<td>400 mg/mL</td>
			<td>6 mL</td>
		</tr>
		<tr>
			<td>Lysine</td>
			<td>0.75 mg/mL</td>
			<td>3 mL</td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>200 mg/mL</td>
			<td>3 mL</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>NA</td>
			<td>q.s.p. 20 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Stock concentrations and volumes of solutions used to produce 20 mL of the gelification mixture. The volume of each stock solution must be proportionally adjusted to produce lower or higher final volumes of the gelification mixture.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reaction Mix Reagent</td>
			<td>Regular mastermix</td>
			<td>Gelification mastermix</td>
		</tr>
		<tr>
			<td>Oligomix (25X)</td>
			<td>1 &#181;L</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>PCR buffer (2X)</td>
			<td>12.5 &#181;L</td>
			<td>12.5 &#181;L</td>
		</tr>
		<tr>
			<td>Gelification Mixture*</td>
			<td>-</td>
			<td>5 &#181;L</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>6.5 &#181;L</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DNA sample*</td>
			<td>5 &#181;L</td>
			<td>5 &#181;L</td>
		</tr>
		<tr>
			<td colspan="3">*maximum of 20% of the final reaction volume</td>
		</tr>
	</tbody>
</table>
Table 2: Volumes of reagents to produce qPCR master mixes for regular or gelified reactions. The difference between the two master mixes is that water is added to the regular master mix whereas the gelification mixture is added (i.e., instead of water) to the gelification master mix.

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

1.5K Views.  Fundação Oswaldo Cruz (Fiocruz). Het gelificatieprotocol produceert gebruiksklare qPCR-reacties die de tijd en stappen verminderen die nodig zijn om een run te starten. Dit protocol maakt het mogelijk om qPCR-reacties te produceren en te controleren op kwaliteit en grote hoeveelheden tegelijk, waardoor de kans op operatorfouten tijdens receptberekeningen of aliquoting in putten wordt verkleind. Deze methode kan worden toegepast op elke qPCR die al volledig is geoptimaliseerd in het conventionele bevroren formaat.Visue...

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

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Genetics

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 ...

Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates

American trypanosomiasis is transmitted to humans by triatomine bugs through the ingestion of contaminated food, by blood transfusions or accidently in hospitals and research laboratories. In addition, the Trypanosoma cruzi infection is transmitted congenitally from a&#160;chagasic mother to her offspring, but the male partner&#39;s contribution to in utero contamination is unknown. The findings of nests and clumps of amastigotes and of trypomastigotes in the theca cells of the ovary, in the goniablasts and in the lumen of seminiferous tubules suggest that T. cruzi infections are sexually transmitted. The research protocol herein presents the results of a family study population showing parasite nuclear DNA in the diploid blood mononuclear cells and in the haploid gametes of human subjects. Thus, three independent biological samples collected one year apart confirmed that T. cruzi infections were sexually transmitted to progeny. Interestingly, the specific T. cruzi antibody was absent in the majority of family progeny that bore immune tolerance to the parasite antigen. Immune tolerance was demonstrated in chicken refractory to T. cruzi after the first week of embryonic growth, and chicks hatched from the flagellate-inoculated eggs were unable to produce the specific antibody.&#160;Moreover, the instillation of the human semen ejaculates intraperitoneally or into the vagina of naive mice yielded T. cruzi amastigotes in the epididymis, seminiferous tubule, vas deferens and uterine tube with an absence of inflammatory reactions in the immune privileged organs of reproduction. The breeding of T. cruzi-infected male and female mice with naive mates resulted in acquisition of the infections, which were later transmitted to the progeny. Therefore, a robust education, information and communication program that involves the population and social organizations is deemed necessary to prevent Chagas disease.

The Trypanosoma cruzi agent of Chagas disease produces long-lasting asymptomatic infections that abruptly develop into clinically recognized pathology. The following research protocol describes a short-run family-based epidemiological study to unravel the T. cruzi infection transmitted sexually from parent to progeny.

American trypanosomiasis is transmitted to humans by triatomine bugs through the ingestion of contaminated food, by blood transfusions or accidently in ...

Immunology and Infection

Quantitation of Rabies Virus in Various Bovine Brain Structures

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major zoonotic risk. Here, our objective was to utilize a laboratory protocol to determine the relative copy number of the rabies virus (RABV) genome in different bovine brain anatomical structures using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR quantifies the specific number of gene copies present in a sample based on fluorescence emitted after amplification that is directly proportional to the amount of target nucleic acid present in the sample. This method is advantageous owing to its short duration, reduced risk of contamination, and potential to detect viral nucleic acids in different samples more easily compared to other techniques. The brains of six rabid animals were divided into six anatomical structures, namely the Ammon&#8217;s horn, cerebellum, cortex, medulla, pons, and thalamus. All brains were identified as positive for RABV antigens based on a direct immunofluorescence test. The same anatomical structures from the brains of four RABV-negative bovines were also assessed. RNA was extracted from each structure and used for qRT-PCR. An assay was performed to determine the copy numbers of RABV genes using an in vitro transcribed nucleoprotein gene. The standard curve used to quantify viral RNA exhibited an efficiency of 100% and linearity of 0.99. Analysis revealed that the cortex, medulla, and thalamus were the ideal CNS portions for use in RABV detection, based on the observation that these structures possessed the highest levels of RABV. The test specificity was 100%. All samples were positive, no false positives were detected. This method can be used to detect RABV in samples that contain low levels of RABV during diagnosis of BPR.

This protocol presents a qRT-PCR-based approach for determining the rabies virus nucleoprotein (N) gene copy number within various bovine brain anatomical structures using in vitro transcription.

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major ...

Chemical Cartography Approaches to Study Trypanosomatid Infection

Pathogen tropism and disease tropism refer to the tissue locations selectively colonized or damaged by pathogens, leading to localized disease symptoms. Human-infective trypanosomatid parasites include Trypanosoma cruzi, the causative agent of Chagas disease; Trypanosoma brucei, the&#160;causative agent of sleeping sickness; and Leishmania species, causative agents of leishmaniasis. Jointly, they affect 20 million people across the globe. These parasites show specific tropism: heart, esophagus, colon for T. cruzi, adipose tissue, pancreas, skin, circulatory system and central nervous system for T. brucei, skin for dermotropic Leishmania strains, and liver, spleen, and bone marrow for viscerotropic Leishmania strains. A spatial perspective is therefore essential to understand trypanosomatid disease pathogenesis. Chemical cartography generates 3D visualizations of small molecule abundance generated via liquid chromatography-mass spectrometry, in comparison to microbiological and immunological parameters. This protocol demonstrates how chemical cartography can be applied to study pathogenic processes during trypanosomatid infection, beginning from systematic tissue sampling and metabolite extraction, followed by liquid chromatography-tandem mass spectrometry data acquisition, and concluding with the generation of 3D maps of metabolite distribution. This method can be used for multiple research questions, such as nutrient requirements for tissue colonization by T. cruzi, T. brucei, or Leishmania, immunometabolism at sites of infection, and the relationship between local tissue metabolic perturbation and clinical disease symptoms, leading to comprehensive insight into trypanosomatid disease pathogenesis.

This protocol describes the steps to generate a 3D model of metabolite distribution during trypanosomatid infection, including sample collection, metabolite extraction, an overview of liquid chromatography-tandem mass spectrometry data acquisition, 3D model generation, and finally, data visualization.

Pathogen tropism and disease tropism refer to the tissue locations selectively colonized or damaged by pathogens, leading to localized disease symptoms. ...

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi (T. cruzi), is an obligate intracellular parasite with a diverse biology that infects several mammalian species, including humans, causing cardiac and digestive pathologies. Reliable detection of T. cruzi in vivo infections has long been needed to understand Chagas disease&#39;s complex biology and accurately evaluate the outcome of treatment regimens. The current protocol demonstrates an integrated pipeline for automated quantification of T. cruzi-infected cells in 3D-reconstructed, cleared organs. Light-sheet fluorescent microscopy allows for accurately visualizing and quantifying of actively proliferating and dormant T. cruzi parasites and immune effector cells in whole organs or tissues. Also, the CUBIC-HistoVision pipeline to obtain uniform labeling of cleared organs with antibodies and nuclear stains was successfully adopted. Tissue clearing coupled with 3D immunostaining provides an unbiased approach to comprehensively evaluate drug treatment protocols, improve the understanding of the cellular organization of T. cruzi-infected tissues, and is expected to advance discoveries related to anti-T. cruzi immune responses, tissue damage, and repair in Chagas disease.

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

The present protocol describes light-sheet fluorescent microscopy and automated software-assisted methods to visualize and precisely quantify proliferating and dormant Trypanosoma cruzi parasites and T cells in intact, cleared organs and tissues. These techniques provide a reliable way to evaluate treatment outcomes and offer new insights into parasite-host interactions.

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Biology

Optimized CRISPR-Based Molecular Diagnostics Protocol for Point-of-Care Use

Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents

Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes that are suitable for use at point-of-care settings such as fast turnaround times for results, convenient simple readouts, and no requirement of complicated instruments. However, the reactions can be cumbersome to perform at the point of care due to their many components and manual handling steps. Herein, we provide a step-by-step, optimized protocol for the robust detection of disease pathogens and genetic markers with recombinase-based isothermal amplification and CRISPR-based reagents, which are premixed and then freeze-dried in easily stored and ready-to-use formats. Premixed, freeze-dried reagents can be rehydrated for immediate use and retain high amplification and detection efficiencies. We also provide a troubleshooting guide for commonly found problems upon preparing and using premixed, freeze-dried reagents for CRISPR-based diagnostics, to make the detection platform more accessible to the wider diagnostic/genetic testing communities.

Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases

Here, we present the step-by-step preparation of premixed, lyophilized recombinase-based isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based reactions, which can be used for the detection of nucleic acid biomarkers of infectious disease pathogens or other genetic markers of interest.

Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes ...

Bioengineering

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.

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 ...

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

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