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

The gelification protocol produces ready to use qPCR reactions that decrease the time and steps required to start a run. This protocol allows qPCR reactions to be produced and controlled for quality and large quantities at once, decreasing the chances of operator error during recipe calculations or aliquoting into wells. This method can be applied to any qPCR that is already fully optimized in the conventional frozen format.

Visual demonstration of this method is important because two of the main quality control steps are visual. To prepare the melezitose solution, weigh four grams of melezitose in a 15 milliliter plastic tube, add six milliliters of nuclease free water, and vortex at maximum speed until the powder is solubilized. Make the final volume to 10 milliliters with nuclease free water, then label and store the solution at two to eight degrees Celsius for up to six months.

To prepare the trehalose solution, weigh four grams of trehalose in a 15 milliliter plastic tube, add six milliliters of nuclease free water and vortex at the maximum speed until the powder is solubilized. Then make up the volume to 10 milliliters with nuclease free water and filter the solution through a 0.2 micron filter. Label and store the solution at two to eight degrees Celsius for up to six months.

To prepare the glycogen solution, weigh two grams of glycogen in a 15 milliliter tube, add six milliliters of nuclease free water, and vortex until the powder is solubilized. Keep the solution at two to eight degrees Celsius for eight to 12 hours because the solubilization of glycogen produces lots of bubbles. The following day, removing the solution from incubation, make up the volume to 10 milliliters with nuclease free water and store the labeled solution at two to eight degrees Celsius for up to six months.

To prepare the lysine solution, weigh 7.5 milligrams of lysine in a 15 milliliter tube, add six milliliters of nuclease free water, and vortex until the powder is solubilized. Make up the volume to 10 milliliters with nuclease free water. Filter the solution through a 0.2 micron filter and store at two to eight degrees Celsius for up to six months.

To prepare the gelification mixture, mix the appropriate volumes of stock solutions in a 50 milliliter plastic tube. Mix the reagents by inverting the tube 10 times and store the labeled solution at two to eight degrees Celsius for up to three months. To prepare the qPCR master mix for gelification, thaw the reagents in a refrigerated container.

Then mix appropriate volumes of the reagents in a 1.5 milliliter tube to prepare enough master mix for an eight tube strip or a 96 well plate. Next pipette 18.5 microliters of the gelification master mix onto each reaction well and place the tubes or plate in the heat conductive support inside the vacuum oven. If using 96 well plates, place one bentonite clay bag in the oven for every two 96 well plates for water absorption.

When the cycle is completed, check the tubes or plates for proper gelification of the reagents by ensuring that the volume is visibly reduced to about five microliters and the liquids do not move upon tapping the tubes or plates with fingers. Seal and store the tubes or plates at two to eight degrees Celsius for eight to 12 hours before use. To use the gelified qPCR, remove the tube strip or plate from the refrigerator and open it in a workstation for sample manipulation.

Add 15 microliters of nuclease free water and five microliters of DNA sample to each reaction vessel. Seal the tubes or plates, run the desired experiment, and perform regular data analysis. In the present protocol, the temperature inside the chamber is kept constant at 30 degrees Celsius, while the pressure varies between 910 to 930 millibar and near vacuum.

After the vacuum cycle, the master mix inside the tube decreases in volume, becomes gelified at the bottom and does not splatter on the walls when the tubes are tapped. If the gelification is incomplete, the liquid spatters on the tube walls when the tubes are tapped. Trypanosoma cruzi-DNA detection using published oligonucleotide sequences is shown here.

Detection of the same sample when gelification was not correctly executed, resulted in a loss of sensitivity. The most critical step is the calculation of reagents volume prior to gelification. The operator must remember to not add water, since it will be removed during the process.

If the protocol steps are followed closely, operators with basic laboratory and molecular biology skills will have no difficulty performing the gelification process.