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

Summary

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.

Abstract

Real-time PCR (qPCR) is a remarkably sensitive and precise technique that allows for amplifying 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 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.

Introduction

Chagas disease was discovered in the early 20^th century in rural regions of Brazil, where poverty was widespread^1,2. 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 food^3,4.

The diagnostic of Chagas disease can be made through the observation of clinical symptoms (especially the Romaña sign), blood smear microscopy, serology, and molecular tests such as real-time PCR (qPCR) or isothermal amplification^4,5,6,7,8,9. 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 failure^6,8,10,11,12. 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 storage^13,14,15.

To circumvent this obstacle, conservation techniques such as lyophilization and gelification have been explored^16,17. 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'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' activity upon rehydration¹⁹. Once stabilized inside the reaction tubes, the reactions can be stored for a few months at 2-8 °C or a few weeks at 21-23 °C instead of the regular -20 °C. This approach has already been incorporated in tests designed to help diagnose diseases such as Chagas disease, malaria, leishmaniasis, tuberculosis, and cyclosporiasis^13,14,15,20.

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.

Protocol

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 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.

1. Melezitose solution
   1. 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.
      ​NOTE: More water can be added to facilitate solubilization, taking care not to exceed the final volume (see below).
   2. Make up the final volume to 10 mL with nuclease-free water. Label and store at 2-8 °C for up to 6 months.
2. Trehalose solution
   1. 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).
   2. Make up the volume to 10 mL with nuclease-free water and filter the solution through a 0.2 µm filter. Label and store at 2-8 °C for up to 6 months.
3. Glycogen solution
   1. 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).
   2. Keep the solution at rest at 2-8 °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 °C for up to 6 months.
4. Lysine solution
   1. 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).
   2. Make up the volume to 10 mL with nuclease-free water and filter the solution through a 0.2 µm filter. Transfer the solution to an amber flask or protect it from light. Label and store at 2-8 °C degrees for up to 6 months.
5. Gelification Mixture (GM)
   1. In a 50 mL plastic tube, mix the volumes of stock solutions according to Table 1.
   2. 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 µm filter before transferring it to an amber flask.
   3. Transfer the solution to an amber flask or protect it from light. Label and store at 2-8 °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 °C.

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).

1. 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 µL containing 5 µL 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).

3. Gelification of the reagents on the reaction vessels

1. Appropriately multiply the volumes shown in Table 2 for preparation of an eight-tube strip or a 96-well plate.
2. Pipet 18.5 µL 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.
3. 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.
4. 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.
5. Subject the tubes/plate containing the gelification master mix to three vacuum cycles (30 ± 5 mBar) of 30 min each, alternating with vacuum release until the atmospheric pressure is achieved (900-930 mBar), under controlled temperature (30 °C ± 1 °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.
6. 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).
7. Seal and store the tubes/plates at 2-8 °C for 8-12 h before use.

4. Using a gelified qPCR

1. Remove the tube strip or plate from the refrigerator and open it in a workstation for sample manipulation. Add 15 µL of nuclease-free water to each reaction vessel.
   NOTE: The volume of gelified reagents is considered to be about 5 µL. So, together with the DNA sample volume (see below), the final reaction volume is 25 µL.
2. Add 5 µL 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.
3. Seal the tubes/plates and proceed to the equipment of choice. Run the experiment and proceed to regular data analysis.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Bentonite clay bags (activated)	Embamat Global Packaging Solutions (Barcelona, Spain)	026157/STD	Not to be confused with silica gel packs
Glycogen	Amersham Bioscience	Cat# US16445
Lysine	Acros Organic	Cat# 365650250
Melezitoze	Sigma-Aldrich	Cat# 63620
Nuclease-free water	preferred vendor
Oligonucleotides	preferred vendor
PCR mastermix	preferred vendor or Instituto de Biologia Molecular do Paraná (IBMP, Curitiba, Brazil)	Chagas NAT kit
PCR thermocycler	preferred vendor
software for vacuum oven	Memmert Gmbh	Celsius v10.0
Trehalose	Sigma-Aldrich	Cat# T9531
Trypanosoma cruzi DNA	from in-house cultivated parasites, or purchased from accredited vendors such as ATCC
Vacuum oven	Memmert Gmbh	VO-400