This method can help answer key questions in the infectious disease field. In regards to Dengue fever, or Chikungunya fever. The main advantage of this technique itself, it's able to quantify viral RNA in a simple and rapid way.
Although this method can provide insight into a basic virus research, it can also be applied to other research such as high through put drug screening study and clinical diagnosis against human populogenic viruses. Major demonstration of this method is critical. As the sample processing stamps and a biosafety cabinet are important to avoid cross contamination or laboratory infection.
Demonstrating the procedure will be an assistant professor and our technical assistant from our laboratory. After preparing the DNA template for the RNA standard, mix the in vivo transcription reaction with 100 nanograms of the prepared DNA template in a 0.2 milliliter PCR tube. Incubate at 37 degrees Celsius in a thermo cycler for two hours.
After this, add one microliter of DNAase and continue the incubation at 37 degrees Celsius for 15 minutes. Set out an RNA purification kit and add 80 microliters of RNAase free water and 350 microliters of capture buffer, including 1%betamarcaptoethanol to the reaction mixture in a 1.5 milliliter tube. Add 250 microliters of 100%ethanol and use a pipette to mix.
Then, assemble an RNA purification column with a collection tube. Transfer the RNA sample to the purification column. Centrifuge the column at 8, 000 times G for 15 seconds and discard the flow through.
Next, apply 500 microliters of washing buffer to the column and centrifuge at 8, 000 times G for two minutes. After this, place the column into a 1.5 milliliter micro centrifuge tube. Add 50 microliters of RNAase free water and incubate at room temperature for a minute.
Centrifuge the column at maximum speed for one minute to allute the RNA. Using a spectrophotometer, measure the optical density of 3 microliters of the alluted RNA at 260 nanometers. Then, determine the copy number of the synthesized Dengue virus three prime UTR RNA.
Store the RNA standard at minus 80 degrees Celsius until ready to use. First, mix 199 microliters of processing buffer with one microliter of nuclease free proteinase K.Using cell culture medium, serially dilute the prepared Dengue virus three prime UTR RNA one to ten to obtain RNA standards at concentrations ranging from 5, 000, 000 to 5, 000 copies per microliter by repeating pipetting and vortex. Transfer 5 microliters of the culture supernatant of Dengue virus infected cells and the Dengue virus 3 prime UTR RNA standard to either A2 PCR strips or the wells of a 96 well PCR plate.
Mix in five microliters of the solution containing processing buffer and proteinase K.Centrifuge briefly at 200 times G for five seconds. Incubate the samples in a thermo cycler at 25 degrees Celsius for ten minutes and then at 75 degrees Celsius for five minutes. Using Dengue virus three prime UTR specific primers and a fluorogenic probe, prepare an RTQ PCR master mix with a one step RT PCR reagent in a 1.5 milliliter micro centrifuge tube.
Alloquote eight microliters of the master mix into a well of a 96 well real time PCR plate. Then, add two microliters of each sample to each well of the 96 well real time PCR plate. Seal the plate with optically clear adhesive film.
Briefly centrifuge the plates at 200 times G for five seconds to remove any air bubbles. Then, place the plate in a real time PCR instrument. Using a real time PCR associated software, navigate to the set up window and assign the well of a reaction to be analyzed as the unknown sample.
Next, assign the well of the serially diluted Dengue virus three prime UTR RNA as the standard and type the expected copy number of RNA standard in each well. Assign the non-template control as the negative control. Then, start the RT PCR instrument and cycle the plate as outlined in the text protocol.
In the analysis window, click on analyze and make sure that the correlation coefficient of the standard curve generated is equivalent to or greater than 0.98. Generally, use the default CT settings for the analysis. In this study, a serial ten-fold dilution of the standard Dengue virus RNA is subjected to a direct RTQ PCR analysis using three prime UTR specific primers and a fluorgenic probe.
The linear curve of this analysis shows that the correlation is good. Next, this direct RTQ PCR assay is applied to quantify the Dengue virus in the culture supernatant of virus infected cells. A good correlation is seen between the Dengue virus infection titer and the CT, a cycle number that is considered to be the point at which the fluorescent signal rises with exponential growth above the background.
When CT values generated from a serial dilution of the Dengue virus stock with known infectious titers are plotted on the previously generated standard curve, the data for the samples ranging between 08 and 8, 000 PFU per reaction are seen to be within the range of those subjected to standard RNA. This indicates that Dengue virus samples with a wide range of infectious titers can be analyzed at the same time when using this technique. This assay is further assessed for its applicability to validating anti-viral agents against Dengue virus.
When treated with 50 micrograms per milliliter of MPA, a reduction of approximately 99.87%of the DMSO treated control culture is observed. Lastly, applications of this assay with other RNA viruses are tested. When a stock of Yellow Fever Virus 17D vaccine strain is subjected to direct RTQ PCR, a standard curve is generated with a good direct correlation between virus titres and CT values.
Likewise, direct RTQ PCR analysis of the measles virus and chikungunya virus raw strain stock gives a good regression between infectious titers and CT values. This technique will pave the way for the researchers in the field of screening study. Allowing a large number of samples to explore new antiviral drugs equals simplistic.
Don't forget that working with re-infectious materials can be extremely hazardous and precautions such as the use of personal protective equipment and a clean biosafety cabinet should always be taken while performing this procedure.
