Episode: 3248
Title: HPR3248: SARS-CoV-2 detection by PCR explanation
Source: https://hub.hackerpublicradio.org/ccdn.php?filename=/eps/hpr3248/hpr3248.mp3
Transcribed: 2025-10-24 19:40:04

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This is Haka Public Radio episode 3248 for when the 13th of January 2021, today's show is entitled,
SARS-CoV-2 Detection IPCR explanation and is part of the series, Health and Health Care.
It is the 30th show of the EASY and is about 24 minutes long and currently in a clean flag.
The summer is, this episode summarizes the process to detect the virus that causes COVID-19 IPCR.
This episode of HPR is brought to you by Ananasthost.com.
Get 15% discount on all shared hosting with the offer code HPR15. That's HPR15.
Better web hosting that's honest and fair at Ananasthost.com.
Hello Haka Public Radio fans, this is B's once again with another episode.
This one is a requested episode to talk about the mechanics of the SARS-CoV-2 PCR test.
Just a little point of clarification, the virus that causes the disease of COVID-19 is called SARS-CoV-2
because it is the second version. What we knew as SARS is the first strain of this type of virus.
For a little bit of background, I'm not going to go too deep into the full background of all of
molecular biology, but to start out with the coronavirus or this particular coronavirus in all,
coronaviruses are a family of viruses called coronaviridae and they are known for having a positive
strand RNA genetic backbone for their inside of a capsid that is made out of protein.
So what positive strand RNA means, that's the type of virus it is, it means that what encodes
its genetic material is not made out of DNA like what are what is made, what we are made out of,
we have RNA obviously as well, but our RNA is a transient chemical in our bodies that only get produced
on the way to producing proteins in a regulated fashion. Like I said, I don't want to go too deep
into all of the biology of it, but the yeah, the central dogma of molecular biology is DNA turns into
RNA, turns into protein, and that's what happens in most living beings on this planet, but
for certain viruses, they store their primary genetic material as RNA, and so positive strand RNA
means that the thing that you are, your genetic code, you know, there's two copies of your DNA,
one goes in one direction, one goes in the other, so it really is like what direction
your the DNA segment is being produced, is it being produced from the, from the reverse version
of the thing that makes the protein or the thing that makes the protein, and the majority of
the really fast killing viruses are in negative strand RNA viruses because they can be
immediately turned into virus, but things like Ebola, for instance, or like that, for
coronavirus, they are positive strand, so before you can, they're really easy at making more
copies of itself, but to make more proteins, it has to first turn itself into the opposite strand,
so that it can make more copies of itself. So that's a little bit about the background. I'm trying
to stay as high off of the biology as I can, because I can get really deep really fast and go
really wide really fast, but for this particular test, to understand it, you first have to understand
a couple things, one is that the genetic material is made out of RNA, and RNA, unlike DNA, is a lot
less stable at room temperature because of that additional hydroxy group on the deoxy, so the
difference between DNA and RNA is deoxyribonucleic acid, and RNA is ribonucleic acid, and so the
difference in their makeup really of the two is one OH group on the ribos part of a nucleoside,
and with that additional group, it makes it a lot more reactive, and so a lot more easily
to break down. And so that's why, for instance, the Pfizer vaccine has to be stored at minus 80,
because the traditional way of storing RNA is at negative 80 degrees, or at least below
negative 60 degrees Celsius, whereas DNA, as you've ever seen in the true crime episode,
it's pretty stable at room temperature for very long periods of time, and when it's completely
dried out, on like a blood stain, it could be years, that it could be stable, at least parts of
the genetic material can be still recoverable. So, next thing, so that's one thing now,
the other thing is the coronavirus attacks specific types of cells in your body, like all viruses,
they're a very organ or organelle specific, or at least tissue type specific,
and so one of the places that they will attack is in your nasopharynx all the way down into your
pulmonary system, and so that's why they have to stick that horrible thing either through your
nose or to the back of your throat, because those are the cells that can be infected by the disease.
If you try to get it out of your finger, for instance, there wouldn't be very much of it there,
because it doesn't want to go inside of finger cells. The proteins that it has the ability to
attach to or found in different types, in the specific types of cells, so what it does is it
goes into those cells, it sticks, it's RNA inside of it, and then I'll share a link on
to the coronavirus Wikipedia page, because it kind of has a good visual there about how it goes
into the cell and attacks the cell, puts its genetic material in there, and forces your body to
make more copies of itself. But first thing we want to do is want to get some of it, and then we
want to be able to detect it. In molecular diagnostics, there are two types usually of detection.
Because the thing that you're trying to detect is so small, and in such small amounts,
you either have to amplify the signal or magnify the target, and PCR uses a target amplification.
So what's the difference between the two? The signal is the thing that you're trying to see.
It is the, let's just say for simplicity sake, it's the color that you're trying to see.
You can either make it so that there's more copies of that color on that one molecule,
or you can make that one molecule brighter, or you can make more copies of that molecule,
so that you can see that very dim, that faint signature that one molecule has. If you have a
million of it, you'll be able to see it a lot more clearly. So those are two different types.
Something like the traditional southern blot that uses, that kind of uses
target, I mean, signal amplification, because you're binding radioactive material to
the DNA, and you can see radioactive material a lot easier than you can see.
You can see, you know, just DNA. On the other hand, something like PCR is target amplification,
because you're making more copies of the target itself. Like I said before, the first step is
collection. When you collect the sample, you either collect it in a special cocktail that's known
as universal transport media, or viral transport media, and it has a chemical agents in it that
both stabilize the genetic material and neutralize the virus. It makes it so that it's more safe to
handle outside of level 2 by a safety cabinet. It's also more expensive, so not all labs use that.
Other labs just use simple PBS, which is phosphate-buffered saline. Yes, just saltwater at a certain pH.
And that also stabilizes it. Stabilizes the nucleic acid, but it does not
inactivate the virus, which means that you should not be handling PBS tubes outside of a,
and opening them outside of a hood. Because when you open it, you can aerosolize the virus,
and it's just like, no, sneezing on someone. At that point, you have aerosolized live virus that
can go and affect people. But in general, most people just stay safe, and whether it's PBS or VTM,
they just process it under a hood. So once you have that first set of material, you have to
extract the nucleic acid and separate it from all the junk and the human cells and everything
that's in there. And it's a process called nucleic acid extraction. Sometimes you are doing a
total RNA extraction. You might be doing a total nucleic acid extraction where you're not separating
between DNA and RNA. And then you use some other mechanism to separate DNA and RNA later.
Because there would not be any copies of this virus in DNA form, or very little of it,
you don't really have to worry about that. So all you do is you do a total nucleic acid extraction,
which there's various different methods and vendors that do that. But one of the most common ways
of doing it is using the chemical makeup of nucleic acid against it itself. And using the
different chemical properties to first get it out of solution, then get rid of any proteins that
might be attached to it because proteins usually encircle the nucleic acids very readily.
And then, so you want to first get all of it, then you want to purify and remove any proteins
that are attached. Then you want to remove the stuff that you use to purify the proteins,
and then you can elute into a buffer, a water-based buffer.
There, like I said, there's a couple of different ways to do it. Sometimes some methods use the
most of the time you're using, you're using things about the charge of
a nucleic acid and using those properties to purify it. Sometimes you're using, when you're doing
like a DNA extraction, you might just be using other things. One easy thing you can do to,
and this is an experiment I've done with my kids and you can do it too, is you can look it up on
the internet to really simple. You can take, because of the way strawberries,
genetic material is they have a really large amount of DNA per cell. And it's really easy to do
your own DNA extraction on strawberries. Basically, what you need is some soap, some salt,
and some isopropyl alcohol. And it's one of the most basic ways of doing a nucleic acid extraction.
You use the soap to disrupt all the salt membranes. And the salt will make the nucleic acid
precipitate out, and then the isopropyl alcohol will make the proteins separate from the salt.
And when you're finishing it with this snot looking gooey material, and it's DNA, and you can show it
to your kids, and they can do it themselves, and they can get the snot, and they can say, I've
purified DNA. It's really cool to see their faces when they see that. So yeah, that's the first
thing you have to do, just purify it. Once it's purified, it's not stable at room temperature. So if
you're not going to proceed to the next steps, you need to store it at minus 80 degrees Celsius.
Usually ultra low freezer that goes anywhere between minus 60 and minus 80.
The next step after that is you have to, and this is usually done on an instrument with a bunch of
proprietary enzymes from the different vendors. But the first thing you have to do is you have to
turn that RNA into DNA in a process called reverse transcription. And if you've ever heard of
HIV, HIV is another type of virus that uses reverse transcription in its life cycle. And that's
just turning RNA into DNA. It's not something that happens in humans normally, but there are enzymes
that you can find in nature that do this process. And then what PCR really is preliminary chain
reaction, I'm going to do a link to that too, is it's a process where you just you find a, you have
a specific target that you're looking for a sequence that you're looking for. And thanks to
researchers out of China, we know what the sequence of SARS-CoV-2 is. If you know what that is,
then you can make a synthetic set of primers and probes to detect it.
Primers are used to amplify the nucleic acid. So once you've turned it from RNA to DNA,
use these primers to attach to specific regions of the DNA that are specific to this virus
and that are different from other viruses or other human genetics. And then a probe
goes in between. So you need one five prime and one three prime primer, which means basically one
primer that goes on, if you put the DNA strand horizontally, one to go on the left side,
one to go on the right side, and then a probe that will match a sequence in the middle.
And what you do is that the five prime end, you'll start to make a copy of, because of special
enzymes, the original way you used to do it back in the day was you actually, it was really expensive
because before we discovered thermophilosacquaticus, it's an extremophile that lives under sea on
on lava vents. Before we discovered that, every time you did the process, you had to add more enzyme
because every time you got to the step where you heat up the DNA to denature it, you kill all the
enzyme, meaning you had to put more in. But when we discovered that there were some creature nature
that lived in such a extreme temperature and still had, obviously, still had a polymerase that
worked, we were able to use that. And so if we ever hear of, for instance, the
TAC path is made by a company local to me, Life Technologies, which is now part of thermo-fisher
scientific, TAC is the name of that, and it stands for that creature, thermophilosacquaticus,
T-A-Q. But yeah, what you do in the process is you have three different temperatures,
you have a temperature for, let me pull mine, don't want to say it wrong,
you have a temperature for a kneeling step, a temperature for the denature step,
which is really high, like 95 degrees Celsius usually, then you have a temperature for a kneeling,
which is letting the primers and probes attach to the thing that you're trying to attach.
And then you have the extension temperature, which is the temperature that you set it to to allow
the polymerase, which is the enzyme that is going to make a copy of the DNA sequence, it allows
it to work. So it goes basically, and these numbers are rough, depending on what exact
sequence you're working with, but it's about 95 degrees Celsius to the denature,
somewhere between like around 35, 40 degrees Celsius to a kneel, and then 72 degrees to extend.
And those are pretty normal temperatures, and every time you go to the denature, you split all
the DNA apart, when you're in kneel, you let the primers and probes sit down on where they
are supposed to go, and then you let it extend, and now those two copies you just turn into four,
and then you do that again, this four turn into eight, and this eight turn into 64, and so on.
So you can see how that type of exponential amplification can lead to a large amount of the
primers and probes being amplified, or the target sequence that you're looking for being amplified.
Now, what the probe does, the probe itself has a reporter part and a quencher part.
The reporter part is something that when you shine a UV light on, it is excited at a certain
wavelength, and when it comes down from an excitation, it makes a color. So it's a fluorophore,
really, and there's usually a couple different colors you can choose from,
but that's the basic idea, and the quencher quenches the reporter when it's in close proximity
to it. So when you do the anneal step, the quencher reporter are really close to each other,
and they're sitting on the ends of the probe, as the rest of the probe is touching the sequence
that it matches. And when the polymerase comes on, it acts like a little Pac-Man, and
choose up all the stuff that's in front of it, as it's making more DNA behind it. And one of the
things that it, when it clips the probe off, it will separate the reporter from the quencher,
and that will then release the light. Only at that time does it release the light.
And then, when it releases the light, you measure it, and then at the next cycle, you measure it
again, at the next cycle, you measure it again, and eventually enough light is found where you start
to be able to see it, and your detector can actually start to pick it up. Now, all this is the
describing a process called real-time PCR, as opposed to endpoint PCR, where an endpoint PCR,
you just amplify everything, and at the end, you try to measure it, and then you can put the
fluorophore or something else in it at the end, and then try to figure out how much if it was there or not.
That method does work. It does have its drawbacks. The standard method for doing most things,
nowadays, are for real-time PCR. And the reason why that's good is because one is you can use
less, it's more reliable. You can, when you're not looking at just endpoint, and you're looking at
every, between every cycle, if it amplified, you were a lot more sure, because you get to see
like a graph of how much it went up, and that it actually did have a positive reaction at all,
versus, you know, we forgot to add stuff to that, to that well. So we don't know if it actually
worked or not. So there's, there's some other reasons why real-time PCR is used now. But when I was
first starting, there are a lot of tests that we were still using endpoint PCR for detection.
But yeah, real-time PCR is what's used for the most part nowadays. And that's pretty much the
extent of it. The thing that differs from test to test is what exactly is being detected. We know
what the sequence is. But sometimes, you know, depending on the virus, and you know, it's
similarity with, for instance, SARS-CoV-1, there might not be a single piece of nucleic acid that
you want to be able to detect that is specific enough. It might still have things that are too,
similar to that. If you only sequence that one, if you only piece out of that one thing,
you're not sure if it's one disease or another. And so different tests will either use different
sequences or different combinations of sequences from different parts of the genetic material
to make sure that it is, it is only specific to SARS-CoV-2. And that's pretty much how it works.
So in summary, first thing you have to do is you have to get a little piece of cells that have
some of the virus on it, usually in your nasal, either coming up through your nose or going
through your mouth, going to the back of your throat. The next thing you have to do is
purify the nucleic acid inside of that. So you put that swab into a transport media and bring
it to the lab, then you purify the nucleic acid. Then you go through the process of amplifying the
nucleic acid while at the same, well, first you do a reverse transcription, which will turn
that DNA, that RNA into DNA, and then you amplify that DNA and start to detect it as it's being
amplified. Then after that, you either know that you have, it has either amplified or has not,
if it has not amplified, you are negative for the test. If it has amplified, you're positive.
And that's it. So I hope you've enjoyed this episode. If you have any other questions,
you can let me know in the comments. So that will be it. This is kind of a long episode for me,
but I hope it explains with enough detail, but not too much where you get too lost.
This has been another episode of Hacker Public Radio. And as always, we say, keep hacking.
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