Name: Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms (Video) | JoVE Journal
Uploaded: 2022-01-20T12:30:00.000+00:00
Duration: 5 min 50 s
Description: Learn a gelification protocol to produce ready-to-use qPCR reactions for Trypanosoma cruzi DNA detection. This method reduces preparation time, minimizes operator error, and enables quality-controlled large-scale production with visual inspection steps.

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5 minutes

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 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><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

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...

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.

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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We describe a high-throughput colorimetric assay measuring β-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.

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

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.

Chemical Cartography Approaches to Study Trypanosomatid Infection

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.

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

To facilitate the rapid and precise detection of Acinetobacter baumannii, we present a protocol that employs Recombinase Polymerase Amplification (RPA) in conjunction with LbaCas12a endonuclease for identifying A. baumannii infections.

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

Here, we describe a simple and quick procedure for detecting DNA from bee pathogens, such as Lotmaria passim and Nosema ceranae, using an amplification-ready cell lysis, recombinase polymerase amplification, and CRISPR/Cas12a assays.

On-site DNA Detection of Trypanosomatid Parasites and Nosema ceranae Through Alkaline Lysis Coupled to RPA/CRISPR/Cas12a System

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.

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

Learn a gelification protocol to produce ready-to-use qPCR reactions for Trypanosoma cruzi DNA detection. This method reduces preparation time, minimizes operator error, and enables quality-controlled large-scale production with visual inspection steps.

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

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