Immunology and Infection
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Summary July 10th, 2019
This real-time RT-PCR using dsDNA intercalating dye is suitable to diagnose lyssavirus infections. The method begins with RNA extracted from rabies suspected ante-mortem or post-mortem samples, detailing master mix preparation, RNA addition, setup of the real-time machine and correct interpretation of results.
Transcript
This real-time RT-PCR is suitable to rapidly diagnose rabies in ante-mortem and post-mortem samples. The Pan-Lyssavirus primers have been optimized to identify all members of the Lyssavirus genus. This is a rapid, sensitive, and closed-tube assay that detects viruses from across the Lyssavirus genus including highly divergent species from clinical specimens.
The OIE has recently accepted molecular assays to report rabies infection. This is particularly important for decomposed specimens which cannot always be diagnosed by virus culture or antigen detection methods. Due to the sensitivity of this assay, good laboratory practice including separation of the different stages is vital to minimize the risk of contamination.
Demonstrating the procedure will be Daisy Jennings, a diagnostician from my laboratory. Start by quantifying RNA with a micro volume spectrophotometer. Ensure the machine is set to RNA and use one to two microliters of molecular grade water to normalize the machine and establish a baseline.
Measure RNA concentration and adjust it to one microgram per microliter. 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 one 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 a 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 L or Beta-Actin. Click on thermo 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 warmup appears, click run now but ensure that the lamp has less than 15 minutes to warm up. When the PCR run has been completed, proceed with data analysis. First, analyze the Lyssavirus amplification plots.
Positive samples display exponential ramps while negative samples have flat amplification plots with no CT values. Next, analyze the Lyssavirus dissociation curve results of the test samples alongside the control samples. A Lyssavirus positive sample will have a melting temperature between 77 and 80 degrees Celsius and overlap with the positive control.
Next, analyze the Beta-Actin amplification plots and dissociation curves comparing with the controls to interpret the overall result. View the text report and use the details to record the CT and TM values obtained for the control RNA in a control card to confirm that the run was successful and that the test sample results can be reported. This protocol is used to demonstrate the sensitivity of the Pan-Lyssavirus RT-PCR on an RNA dilution series of a control standard virus.
The amplification curve indicates that as little as 10 picograms of Lyssavirus can be detected and the dissociation curves verify the melting temperature of the product. The melting temperature is used to confirm that the amplicon is Lyssavirus specific. The results here demonstrate a melting temperature range across the Lyssavirus genus of 77.34 to 79.67 degrees Celsius.
Melting temperature values below 76.8 or above 80.2 are considered non-specific. The sensitivity of this RT-PCR assay is also determined by detecting RNA from three Lyssavirus positive brain samples. As little as 0.1 picograms per microliter of target RNA can be detected for two out of the three samples.
Notably, representatives from all recognized Lyssavirus species are detected using this assay. If analyzing RNA extracts from clinical samples such as saliva or CSF, Beta-Actin results may be negative due to a lack of host nucleic acids. The addition of an exogenous control may resolve this issue.
Sequencing of Lyssavirus positive samples is recommended to provide additional information regarding the geographic and host origins of a rabies infection. Handling of Lyssavirus positive or suspected positive samples must be according to local health and safety guidelines. The extracted RNA is non-infectious, therefore can be handled within low containment laboratories.
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