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Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR
Chapters
Summary November 10th, 2018
This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.
Transcript
Welcome everyone. My name is Win Surachetpong. I'm an Assistant Professor in Veterinary Microbiology and Immunology Faculty of Veterinary Medicine at Kasetstart University, Thailand.
Me and my team are specializing in emerging viral disease in tilapia. We are now working on tilapia lake virus which cause high mortality in tilapia. Since tilapia is the second most important fish species that is culture worldwide, it provide protein source for a million people and play important role for food security.
So the recent outbreak of tilapia lake virus cause socioeconomic impact that led many scientists to try to find a solution for this problem. So far we need a very highly sensitive, rapid, and accurate method to detect the virus. In this video, we will show you step-by-step to detect tilapia lake virus in fish tissue starting from sample collection, sample population, and real time PCR analyzing.
First, solubilize the overdose of cloth oil in water to euthanize the fish. Place the dead fish on a tray, sterilize the equipment using 95%ethanol, then burned with an alcohol burner. Slowly cut open the fish from the lower abdomen upward, to collect the liver.
Collect part of the liver into a 1.5 milliliter centrifuge tube. The first part of the RNA extraction should be performed in a Laminar Flow Hood, and wear protective equipment. Add one milliliter of RNA extraction reagent into the tube.
Pulverize the liver using a tissue pestle into homogeneous. Add 200 microliters of chloroform into the tube. Then, gently mix by inverting the tube several times.
Set aside for three minutes at room temperature. Centrifuge at four degree celsius at 12, 000 RCF for 15 minutes. Carefully collect the tubes from the centrifuge.
Gently transfer 500 microliters of the colorless upper aqueous phase into a new tube. Add one volume of isopropanol into the tube. Heat at minus 20 degree celsius for two hours or up to overnight to precipitate the RNA.
After the incubation, centrifuge the solution at the same centrifuge condition. Collect the tubes from the centrifuge, then discard the supernatant. The RNA pellet can be seen as pointed by the arrow.
Add one milliliter of 75%ethanol into the tube to wash the RNA pellet, and invert several times. Centrifuge at four degrees celsius, at 10, 000 RCF for 15 minutes. Collect the tubes from the centrifuge, then discard the supernatant.
Draw out the leftover ethanol using the auto pipet, and air dry at room temperature for five to 10 minutes. Add 30 to 60 microliters RNase free water, pre warmed to 55 to 60 degrees celsius to solubilize the RNA pellet. Use one to two microliters of RNA solution to measure the concentration using a micro volume spectrophotometer.
The results will show up on the screen. Dilute the concentration to 200 nanograms per microliter using RNase free water. Perform cDNA synthesis using the chemicals and conditions listed as follows.
Use a thermocycler to convert RNA into cDNA with suggested conditions. The following are the chemicals and conditions used for determining the TiLV load using real time PCR. Dispense six milliliters of the qPCR master mix into each vial of white qPCR strip tube.
Subsequently, four milliliters of cDNA sample is loaded into the well. At this stage a new pipe tip must be changed in every single well, to avoid contamination from sample to sample. Seal the qPCR strip tightly with a transparent qPCR strip cap.
Gently mix the solution by flicking at a tip of the strip. Then spin down using micro centrifuge to collect all the liquid at the bottom of the vessels. Use the two step condition as shown in the video, select the well in 96 well plate that you're going to use.
Select cyber green as the flora for dye. Select unknown as the sample type, and insert a name into a sample name box. Open the lid of a real time PCR machine and place the qPCR strip into the assigned well.
Then close the lid. Run the machine with the selected condition. The machine will start and run after the lid had reached the desired temperature.
After the end of qPCR conditions, amplification curve is shown. Here, the rate arrow points out where the threshold level cuts off between the exponential phase and linear phase which results in CT value. The CT value is then extrapolated into virus log copy number to calculate a number of virus remains using the standard curve.
The melting peak is also displayed to identify whether the virus detected by qPCR is, indeed, TiLV in which the melt peaks generally range between 79.5 and 80.5 degrees celsius. So we hope that this method will be important tools for farmers and organization around the world, who may require implement a control and limit the spreading of TiLV infection.
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