This article discusses the use of real-time quantitative reverse transcription PCR (qRT-PCR) for identifying viral infections. The method involves amplifying viral RNA to detect the presence of specific viruses in samples.
Real-time quantitative reverse transcription PCR is useful for identifying viral infections.
Begin with a sample containing the target viral RNA. Add target-specific primers — aiding in both DNA synthesis and amplification — along with reverse transcriptase enzyme and dNTPs.
Add thermostable DNA polymerase — an enzyme for amplifying DNA across PCR cycles — and SYBR green — a dye that intercalates into double-stranded DNA, dsDNA. Place the reaction in a thermal cycler. Set an appropriate temperature for the primers to bind to the target RNA.
The reverse transcriptase adds dNTPs to the primer while simultaneously cleaving the template RNA, synthesizing cDNA.
Increase the temperature, inactivating the reverse transcriptase. Lower the temperature, allowing the primers to anneal to the cDNA for subsequent amplification. Increase the temperature to reach the extension step, where the DNA polymerase extends the primers, synthesizing dsDNA.
Consecutive cycles result in exponential dsDNA amplification. SYBR green binds to the DNA in increasing numbers, causing a proportional increase in fluorescence emission. Plot the fluorescence signal detected across the cycles.
A positive amplification curve indicates the presence of viral RNA in the sample.
Increase the temperature incrementally to reach the melting temperature, denaturing the dsDNA into single strands. The bound dye molecules get released — losing their fluorescence. Plot the decreasing fluorescence against the temperature.
A peak corresponding to the virus-specific melting temperature confirms viral infection.
Plan the layout of your PCR plate with a spreadsheet, taking into account both test and control samples. Prepare a PCR workstation by disinfecting surfaces, and if using a UV cabinet, turn on the UV light 10 minutes prior to starting.
Remove reagents and primers from the freezer to thaw, but keep the enzyme mix on ice at all times. Mix the reagents, and centrifuge briefly to collect the liquid. Prepare PCR master mixes for lyssavirus and beta-actin, according to the manuscript directions. Leave the master-mixes on ice until ready to use. Mix and centrifuge the prepared master-mixes, and dispense 19 microliters into the relevant wells of the PCR machine-compatible plate or tube strip.
In a separate room or a UV cabinet, carefully add 1 microliter of the prepared RNA. If using a UV cabinet, turn on the UV light 10 minutes prior to starting. After the test samples, add the positive control and the no template control.
When adding the RNA to the master mixes, ensure that it's added to the correct well. Use the plate plan to aid this step.
Seal the plate, checking that the lids are flat across the plate, and spin it down to collect all liquid at the bottom of the wells. Ensure each well has the same volume of liquid and no bubbles are visible. Then, transfer the samples to the PCR machine.
Open the program, choosing 'SYBR Green' with 'Dissociation.' Select the wells to be analyzed, choosing "Unknown" as the sample type and "SYBR" as the fluorescent dye. Label the wells on the plate layout with the sample information, including whether the assay is for Lyssavirus well or beta-actin.
Click on "Thermal Profile Setup" and modify the thermal cycling conditions as specified in the manuscript. Click "Start," then, choose a location to save the file, and check the box to switch off the lamp at the end of the run. When the option to start before lamp warm-up appears, click "Run Now," but ensure that the lamp has less than 15 minutes to warm up.
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