How do they actually perform the RT-PCR tests?


How do they actually perform the RT-PCR tests?

In: Biology

They basically throw in Primers that can attach to the RNA of Coronavirus together with a bunch of materials needed to build new RNA.

If the coronavirus RNA is present the the primers will attach to it and new RNA will be formed. That’s how you test positive.

If there is no amplification (no new RNA created) that means you have no coronavirus RNA in you and you are clean.

The other commenter went into how they find the test results scientifically.

I’ll talk about what it’s like to administer/receive a test.

They have you clear out your nostrils – just blow your nose super hard. Then they insert a long cotton swab into each side of your nose, all the way back to the sinus cavity, and wiggle around to get tissue. It’s about ten seconds on each side.

Then they bag the swab and send it through the process the other person talked about.

[Here]( is a video of someone being tested at a drive-up facility,so you can see what it’s like. [Here’s]( an image of how deep the swab goes.

You get a sample. You digest it/lyse cells (breaking apart membranes and release the inside of cells including nucleic acids like DNA and RNA). You add a DNAse enzyme, which breaks down all DNA in the sample. You follow any established protocol for RNA isolation, where you get rid of proteins and lipids and all and just purify all the RNA in the sample. You take this RNA and conduct RT on it. Meaning, you add a cocktail of enzyme (reverse transcriptase which makes DNA from RNA), random hexamers (these are short sequences called primers that are random in sequence themselves, meaning they complementarily bind all sequences), some buffer to facilitate the reaction, some bunch of DNA nucleotides (these are the building blocks of DNA), and some other things. You run an RT which stands for reverse transcription.You essentially convert all RNA in this sample to its complementary DNA, called cDNA. You don’t amplify, you just reverse transcribe. Then you take this cDNA sample and run another reaction, just called a PCR. You add two primers, one that has a particular sequence binding only to the cDNA coming from the viral RNA, and the other just runs in the other direction some nucleotides away. In this reaction, you have a polymerase and all the reagents and resources you need for a PCR reaction. The primers bind to the cDNA from the virus if it is there, and the polymerase starts copying the DNA making a new Strand. As you have two on opposite sides of the DNA, you eventually starts exponentially amplifying one particular region, the amplicon. As you know where your primers bind and you know the genome of the virus, you know that length of this resultant amplicon. After running this reaction for 40 or whatever cycles, you’ve made an enormous amount of amplicon (assuming it’s there). Then you take this sample and you load it on a gel. The gel has a matrix, it’s agarose. You apply electricity to the gel that is put in a buffer, now you have one side positive one side negative. DNA is negative, so it will be pulled to the positive side. The longer the DNA in your sample, the slower it travels. You also have a dye in the gel that allows you to see DNA under UV light. Anyway, after you run the gel (which also has a well loaded with ladder, which is a cocktail of DNA of known lengths so you know what each spot corresponds to in size. Then you visualize the gel under UV, if you amplified that DNA from the virus, you see an intense band at that particular size (parallel to the ladder). If you don’t see a band there, then there was no virus in the original sample.

Edit: I just described RT-PCR and how we do it in the lab for research. There is of course a more expensive approach called qRT-PCR (stands for quantitative real time reverse transcription polymerase chain reaction). Surprisingly I think that’s the method the CDC is using. The difference here is that after you make cDNA, you don’t do a simple PCR. You add primers and all just the same but you actually also add probes. This is a little beyond ELI5, but probes are also sequences but they bind in between the primers and they fluorescence functioning as a sensor. You have a detector that measures light intensity in that wavelength and so you can tell exactly how much DNA of that sequence is in your sample. I think that’s overkill for a simple yes no test, but I guess that’s how they do it, unless I’m missing something.

It’s a long and complicated procedure but basically you can take a single sample dna/rna from a virus/bacteria cell and they replicate and amplify it enough to be compared to a known bacteria or virus dna.

They extract it, purify it, amplify it and then compare it. Every time they amplify it, the amount doubles. After a few cycles they can go from 1 dna to over a billion in a few hours.

When they compare it, it’s like the “day time talk shows” where they are trying to find the baby daddy.