the information provided in this video such as the primer sequences kits used and thermocycler conditions are from published papers please refer to the accompanying PDF for web links and the full protocol including specific volumes you my name is Lindsay Alton and I'm a postdoctoral research associate for University College London and the pandora ID net consortium this video shows the method for reverse transcriptase pcr which can be used to diagnose the 2019 novel coronavirus this method utilizes the viruses nucleic acid and therefore can be used to diagnose it in both humans and animals before you can do PCR on your samples you will need to extract the virus's RNA biosafety level 3 or BSL three conditions including a cast one or a class to microbiological safety cabinet must be used when handling 2019 and Co V as there is evidence that it can be transmitted from human to human you must follow your local bsl-3 health and safety rules at all times during these procedures you must have a clean chain of laboratories to prevent contamination of your PCR samples there are lots of kits that can be used to extract viral RNA but the kit used here is the direct sole RNA miniprep kit in a new rnas free screwcap tube at 80 microliters of your sample and 240 microliters so three volumes of trayzell mix this thoroughly for five minutes to lies your sample you at an equal volume of 95 to 100% ethanol to your lized sample and mix thoroughly transfer the lized sample into a spin column which is placed within a collection tube you carefully wipe down the spin columns and collection tubes with disinfectant before placing them into an enclosed microcentrifuge bucket to contain any aerosols produced wipe down the outside of the bucket and your gloves with disinfectant following your local safety rules regarding disinfection remove it from the biological safety cabinet place into a micro centrifuge and spin at ten to sixteen thousand G for 30 seconds you remove the bucket from the microcentrifuge and place back into the microbiological safety cabinet before opening the bucket transfer the column into a new collection tube at 400 microliters of direct soul RNA pre-wash to the column and centrifuge as described before discard the flow-through and repeat this step one more time a recommended extra step is to DNA's treat your samples as described in the accompanying PDF protocol you at seven hundred microliters of RNA wash buffer to the column and centrifuge as described before for two minutes rather than 30 seconds to ensure complete removal of all the wash buffer transfer the column carefully to an RNAs free tube to elute your RNA add 50 microliters of DNA's and RNAs free water directly to the column matrix and centrifuge as described before alternatively for a higher concentrated RNA eluted into 25 microliters instead of 50 transfer the elated RNA into a screw cap tube the eluted RNA can be used immediately or stored at minus 70 degrees ensure that you keep track of each of your samples throughout this process by labeling them clearly you you as good practice you should check that your extraction method has worked by quantifying your eluted RNA there are a number of ways to do this which are explained in the accompanying protocol PDF it is important that your pcr samples do not get cross contaminated with other samples in your laboratory to ensure this you need a clean chain of laboratories meaning that your PCR preparation area is separate from your PCR thermo cycler area where large amounts of DNA amplicons are generated ideally you should prepare your pcre agents in a cabinet to protect them from contamination as the polymerase chain reaction requires DNA we need to use reverse transcriptase or RT to create complementary or c DNA strands from the extracted RNA in this protocol we are using one-step real-time rt-pcr where the cDNA is synthesized by a reverse transcriptase in the same tube as the PCR reaction for this PCR we need nuclease free water two times reverse transcriptase PCR buffer a forward and reverse primer plus probe for 2019 and Co V plus 25 times rt-pcr enzyme mix to create a master mix before starting your PCR you will need to order primers and probes specific to the 2019 nkv as described in the literature usually primers will be delivered to you as a lie of relized powder and they will need to be reconstituted in nucleus free water you should receive a data sheet with your primers telling you what volumes are needed to make stock solutions ensure that you have thoroughly mix the water with the powder and before opening your tube spin it down in a centrifuge for more information about fluorescent probes please refer to the accompanying PDF protocol it is advisable to then aliquot some of this stock solution into micro tubes of working solution and freeze them at minus 20 degrees to minimize freeze thering and contamination of your main stock to create your master mix you will need to add the reagents to a separate rnas free tube in this example we have extracted the RNA from three patient samples these are labeled one two and three here the volumes you will need depend on the amount of samples that you are processing in this example we are repeating each of our three samples three times as well as having three positive controls and four negative controls please refer to the supplementary protocol PDF for the PCR reagent volumes needed as well as an explanation about positive and negative controls mix the master mix well by pipetting or gently flicking the tube once you have created your master mix aliquot it into the PCR tubes these may differ or be plates with wells depending on the PCR machine that you are using don't forget to add nuclease free water to your negative controls and 2019 and Kobi nucleic acid to your positive control tubes if you do not have 2019 and Co V RNA for your positive control refer to the supplementary protocol PDF add your extracted RNA samples to the tubes ensuring that you have made a note of the order in which you add them so that you can keep a track of them when you place them in the PCR machine if using tubes ensure you carefully but firmly place the lids on top it is helpful to label the lid so you can keep track of the samples you can now proceed to your PCR amplification laboratory this area should only be used for amplifying DNA and not preparing PCR reagents in this video we are showing you how to set up the conditions for a Corbett research real-time PCR thermo cycler rotor gene but yours may be different please refer to the manufacturers guidelines for how to set up your thermo cycler place the tubes into the rotor the rotor is numbered to ensure that your samples do not get confused once you have placed the silver locking ring on top of the rotor place it into the thermo cycler lining it up with the pin and press it down until you hear it click close the lid of the thermo cycler and then open the thermo cycler program on the computer open a new run in the program on this machine we have to confirm the type of rotor and that we have attached the locking ring then set up the cycle settings the paper we have referred to states that the cycle should be held at 50 degrees for 15 minutes 95 degrees for three minutes and then forty five cycles of 95 degrees for 15 seconds and sixty degrees for thirty seconds ensure that you are acquiring fluorescence in the same channel as your probe in this case we have used SCI five and therefore must detect crimson refer to the accompanying PDF protocol for more information on probes once the conditions are set up run the cycle and save the results to an appropriate folder you can then label each of your samples in the program once the thermocycler has finished you can open the file with your results click on analysis and then on the crimson channel which brings up the PCR cycle curves you can then set and move the threshold or baseline the threshold is defined as the level of detection or the point at which a reaction reaches a fluorescent intensity above background levels ie your negative controls which are shown in black here please note that this is not a quantitative PCR we are purely identifying where the 2019 nkv RNA is present in a sample or not to validate this PCR for the first time if you do not already have a 2019 Enco be positive control you should sequence your positive samples for more information on analyzing PCR results refer to the accompanying protocol PDF you the protocol in this video uses specific kits and equipment to identify 2019 n Co V nucleic acid in samples if you need more information about diagnosing this virus please contact the Pandora ID net or consult further sources such as the Global Health Networks 2019 n Co V knowledge hub or the WH o s website both of which are referenced in the accompanying protocol PDF you you