Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

The overall goal of the following experiment is to use quantitative recombinase polymerase simplification to quantify the DNA concentration of unknown samples. This is achieved by first adding the target DNA internal positive control, DNA primers and fluorescently labeled probes to the reaction. As a second step, the reactions are placed in the real-time PCR machine, which heats the reactions to activate the enzymes and monitors the fluorescence of the probes to detect the generation of target and control amplicons.

Next, the fluorescent data is analyzed using a script to generate the standard curve and to validate the assay. The results show that DNA samples can be quantified accurately within one order of magnitude of the correct concentration based on the validation experiments used to quantify an HIV one target DNA. The main advantage of this technique over existing methods like real-time quantitative PCR, is that RPA is isothermal, so an expensive thermal cycler is not needed.

RPA also requires a lower amplification to temperature, is tolerant to dirty samples, amplifies target to detectable levels within minutes, and utilizes lly enzymes to allow easy transportation and storage at room temperature. To begin clean all bench top and equipment surfaces with a solution of 50%bleach to create a clean pre amplification workspace, remove the 280 millimolar magnesium acetate solution, the rehydration buffer, and the reaction pellets from the minus 20 degrees Celsius freezer and thaw the solutions at room temperature. Then assemble a master mix by transferring 383.5 microliters of the rehydration buffer to a new micro fuge tube at 41.6 microliters of nuclease free water to the buffer and vortex to mix at 35 microliters of magnesium acetate to a separate tube.

Return the magnesium acetate in the rehydration buffer stock solutions to the freezer. Then obtain the primer and prob quas from the four degree Celsius refrigerator. Place them in the pre amplification workspace and turn off the lights to minimize light exposure at 27.3 microliters of each of the 10 micromolar forward and reverse primers to the master mix.

Next at 7.8 microliters of the 10 micromolar hex labeled HIV 1D NA.Probe to the master mix and vortex the tube. Then return the primer and the probe stock solutions to storage reagents for the internal positive control can be added here as indicated in the text protocol to assemble the QRPA reactions. First place the freeze dried enzyme pellets into individual tubes of two, low-rise eight well PCR tube strips pipette 37.5 microliters of the master mix into a tube containing a pellet.

Gently stir the contents of the tube with the pipette tip to dissolve the pellet without creating bubbles. Remove the tip from the reaction carefully to prevent a loss of volume and to make sure to change pipette tips between each tube. Transfer the tubes to a chilled 96 well cold block for at least five minutes to cool the master mix.

In the meantime, load the thermal cycler software with the protocol and the plate layout is indicated in the text protocol. Next, cut two strips of clear plastic micros seal adhesives so that they are slightly wider than the PCR tubes. And also obtain two flat PCR tube strip lids, aliquot the HIV one plasmid template as indicated in the text protocol.

And then add 10 microliters of the template to the appropriate PCR tube. Again, gently stir the mix with the pipette tip. Next, add 2.5 microliters of the magnesium acetate to each tube cap, and then gently place the caps on top of the reaction tubes so that they are not fully sealed.

The magnesium acetate should be clearly visible through the cap. Place the PCR tube strips into a micro centrifuge and centrifuge the tubes for 10 seconds to collect all of the liquid in the bottom of the tube and to remove any bubbles. Combining the magnesium acetate solution with the master mix initiates the QRPA reaction.

Quickly remove the PCR tubes to the cold block to halt the reaction. Next, slowly remove the lids to prevent contaminating the reactions and seal the tubes with a clear micros seal film. Transfer the tubes to the thermal cycler according to the locations in the plate layout.

Close the lid of the thermal cycler and click start run. Make sure to designate a file name for the experiment when the run is finished. View the raw fluorescence data and export it to a spreadsheet which will create a file called quantification amplification results.

To build the standard curve. First, download the standard curve script, then open MATLAB or a similar data analysis program. Open the script and press run to run the script.

Type in the number of data files to be analyzed when prompted. Next, type the number of samples and the number of replicates in each training file. In the command window.

Type in the lowest DNA concentration in log-based 10 copies that was used to build the standard curve and press enter. In the example here. The lowest concentration is one log based 10 copies.

Then type the difference in concentration between each DNA standard into the command prompt and press enter here. The concentration interval is one log based 10 copies. Next, enter the value for the slope threshold into the command window and press enter.

This value is typically between three and five. Type in the number standard deviations above the background for positive threshold, also known as Z, and then press enter typical Z values range from one to five. Next, specify the equipment that was used to collect the data in this case.

Type one for the thermal cycler, and then press enter. If there is an internal positive control, select the default or a new value for the threshold by typing Y or N depending on whether a new threshold is necessary. Finally, select all of the spreadsheet files that will be used to build the standard curve.

The script will then automatically import and analyze all of the data. Realtime QRPA was performed on duplicate samples containing different copy numbers of HIV 1D NA.The onset of detectable amplification indicated by an increase in fluorescence occurs earlier for samples with higher DNA copy numbers than for samples with lower copy numbers. The raw fluorescence data were used to generate a standard curve from amplification of known concentrations of HIV 1D NA.These data were fit to an exponential curve, and the R squared value indicates a high goodness of fit.

The standard curve was used to predict the concentrations of additional DNA samples of known concentration. Adjusting the value of Z to one allows accurate prediction of low DNA concentrations. In contrast, high DNA concentrations are better predicted with a Z value of five.

When attempting this procedure, it's important to remember that RPA lacks true cycles to limit the rate of DNA amplification. So the rate of amplification must be precisely controlled. Consistency between experiments is crucial, so be sure to use the same primer aliquots for all experiments.

Protect reaction components from heat and light. Add magnesium acetate immediately before data collection begins, and keep reactions in the cold block until beginning. The experiment.