When it comes to COVID tests, there are 2 main types – “PCR tests” and “antigen tests.” PCR tests look for evidence of the viral genetic information by trying to make lots of DNA copies of a region of it and seeing if copies get made. Because you’re making copies, it’s super sensitive (sometimes too sensitive for your purposes). Antigen tests look for viral proteins, typically the Nucleocapsid (N) protein. They don’t make copies, so they’re less sensitive but are typically faster and can be done at home. Here’s more of the biochemistry behind them… (text adapted from past posts). 

PCR tests

SARS-CoV-2 is an RNA virus – instead of storing its genetic blueprint (genome) in DNA like we do, it keeps it in RNA. And as single strands. ⠀ 

Within this RNA are instructions for making proteins that the virus needs. A virus really only “cares” about one thing – making more copies of itself and infecting more cells, and so the genes you find in its RNA reflect what it needs to do this. A few examples:⠀ 

* It has to make copies of its RNA, so it needs an RNA-dependent RNA polymerase (RdRP) that can travel along the RNA strand and use it as a template for making a complementary RNA strand – which can then be used to make a copy of the original template strand which can then be used to make a copy of the complementary strand which… you get the point – thanks to the complementary base-pair-ing nature of RNA & DNA (the letter A binds to U (or T in DNA) and G to C) it’s “copyable” And, since the virus encodes for an RNA-dependent RNA polymerase, it’s able to copy its genome. ⠀ 

* Then it has to coat those RNA copies in a protein “shell” called a nucleocapsid for protection on its journey, so it needs a gene for the nucleocapsid protein (this is called the N gene and it is what the CDC tests look for).⠀ 

* Next it has to be able to bud out of the cells its currently in so that it can go find and get into a new cell where it can do it all again, so it needs an envelope protein for this (WHO’s tests look for this “E gene”).⠀ 

* But, before it can get into a new cell, the virus needs to “stick” to that cell’s surface and it does this using Spike (S) proteins that jut out from the viral envelope like a crown (hence the name corona) and bind to receptors on the host-cell-to-be’s surface.⠀ 

Humans don’t need those things for ourselves, so we don’t have instructions for them in our genome, so if scientists find those RNAs in a person, it indicates that a virus is in that person. ⠀ 

But how do you go about finding that RNA? There are a couple of challenges, the first of which is that, even if there’s a bunch of virus, the total amount of RNA is still pretty tiny. And, to make things even worse, RNA is pretty unstable – so, in the process of isolating it (RNA extraction) you lose some of what you started with⠀ 

So you need really sensitive methods to detect it amongst all the other RNA & DNA (collectively called “nucleic acids”) present. And one way to do this is to amplify the signal while not changing the “noise” – make a bunch of copies specifically of the viral genetic info that’s present. ⠀ 

Thankfully, scientists have known for half a century or so how to get a DNA Polymerase to make lots of copies of specific stretches of DNA in vitro (in a tube) using a method called Polymerase Chain Reaction (PCR). The stretches to be copied (amplicons) are specified by short pieces of DNA called “primers” that are designed to bind to where you want the copying to start and stop (one primer per strand). PCR is carried out in a series of cycles where you ANNEAL – bind the primers -> ELONGATE – let the DNA Pol copy the stretch to give you double-stranded DNA -> MELT – raise the temperature so the strands “unzip” and you can do it all again⠀ 

But the DNA Pols we use are DNA-dependent DNA polymerases – a mouthful of a phrase that just means that they make DNA copies from DNA templates. Which brings us to problem number 2: SARS-Cov-2 is an RNA virus?!⠀ 

Thankfully, we have a solution to this too, which is where the “RT” part of “RT-PCR” comes in. RT stands for Reverse Transcription, and it’s the process of making a DNA copy of an RNA template. It’s called reverse transcription because the process of going from DNA to RNA is called transcription, which is what our cells do to make RNA copies of our DNA genes to use as protein-making instructions⠀ 

So, before the actual PCR part, can use a “Reverse Transcriptase” enzyme to make complementary DNA copies of the viral RNA, and now we have DNA that DNA Pol will happily copy if we provide primers.⠀ 

But we still need a way to detect the copies that get made. This is done using fluorescent probes, which are kinda like primers in that they’re short DNA pieces that pair specifically with the region you’re interested in. But instead of binding the ends, they bind somewhere in the middle of the copied region and allow you to see that a copy’s been made. How it works is pretty cool, so if you stick around later, I’ll give some more details. ⠀ 

The primer/probe combos allow us to look for those little stretches of letters in the viral genome that this virus has but we don’t (such as parts of the N, E, or RdRP genes). But in order to find those little stretches on the viral RNA we first need to find the viral RNA. Where do we look for that? Tests are normally performed on swabs from your nose or throat (officially referred to as nasopharyngeal (NP) or oropharyngeal (OP) specimens, respectively or now from the front of your nose (not that huge q-tips). You might even just have to spit.  

Many tests use “TaqMan” probes. These “dual-labeled hydrolysis probes” work using something called FRET. Don’t fret if you don’t know what that means – let me explain. FRET stands for Forster Resonance Energy Transfer and it’s this cool phenomenon that allow you to tell if 2 molecules are nearby each other. One molecule, the “fluorophore” is able to give off light, but only if the other molecule, the “quencher” isn’t nearby.⠀ 

And, in the probes, they *are* nearby (at least in the beginning…) And speaking of beginning in a different sense, the fluorophore (a chemical group called FAM (6-carboxyfluorescein) in the CDC probes) is on the beginning of the probe (what we call the 5’ end) and the quencher (BHQ (Black Hole Quencher) in the CDC probes) is on the other end of the probe (the 3’ end). The probes are only about 20 letters long, so the quencher is near enough to the fluorophore to keep it from shining (they’re just a few nm apart, which is about 100,000 times less than a hair width apart).⠀ 

Light is a form of energy and different wavelengths of light have different energies. If you have a fluorophore and you shine light of a wavelength that the fluorophore “likes,” the fluorophore absorbs the light and enters an “excited state” – but it’s hard to stay excited for long, so it comes down from the high & releases that energy which it had absorbed as light that you can detect.⠀ 

But light isn’t the only way energy can be transferred – another way is through FRET. If the quencher likes the amount of energy the fluorophore would normally give off *and* that quencher and fluorophore are close enough, the quencher can absorb the energy that the fluorophore would normally give off as light – it “quenches” the fluorescence http://bit.ly/2m5hpfh ⠀ 

But in order for that quenching to happen, the fluorophore & quencher have to be close together. When they get separated, the reporter’s free to shine. ⠀ 

And they get separated when the DNA they’re bound to gets copied, because the DNA Pol used to do the copying, Taq polymerase, has 5’ nuclease activity. So, if it goes to copy some DNA and there’s a probe “road block,” Taq can “chew up” the probe when it runs into it – this allows Taq to displace the probe and carry out its copying – and it separates the fluorophore from the quencher, allowing us to see the light. ⠀ 

And this light serves as a signal that a copy got made since Taq can “only” run into the probe if it’s copying the DNA the probe’s bound to (I mean theoretically they could just bump into each other anywhere but it’s really unlikely unless they’re brought together by the copying process). And a copy getting made means that the target sequence (such as the viral gene) was present to get copied, indicating infection. ⠀ 

But, since you have so few copies in the beginning, the amount of fluorescence you originally see is really low – indistinguishable from the background “noise” – so we need to amplify it.  ⠀ 

In between each copying cycle, you have another melt step where the strands come apart, and this allows unchewed (quenched) probe to bind during the anneal step (the primers also bind then too). And then when you enter the elongation phase, where the DNA gets copied, the Taq runs into and chews up more probe. And so more dye gets “un-quenched” so you see more fluorescence. ⠀ 

Since each strand is used to make 1 copy each cycle, you go from 2 -> 4-> 8 -> 16 -> 32, etc. (exponential growth) so you see an exponential increase in fluorescence until something runs out (probe, primer, nucleotides, etc.). The more copies you start with, the faster the fluorescence will climb, so you can designate a “reference threshold” level of fluorescence and then see “how fast” different sample/primer/probe combos reach that reference. By “how fast,” we usually talk in terms of amplification cycles.⠀ 

If you plot cycle # vs fluorescence, you get a sideways-candy-cane shaped curve, which there isn’t really a good symbol combo for, but kinda ,- ish (just look at the pic…)⠀ 

What you’re looking for is a value called the Ct value which is the # of cycles it takes to pass a “threshold line” corresponding to the background fluorescence level (noise) – when you cross the threshold it means you get “above background” – so you know you have real signal and not just noise. It’s hard to tell in the first cycles because you have so little being copied, but the more copies are in your initial “little amount” the sooner (fewer cycles) you’ll cross the threshold (lower Ct values). Ct values are therefore sometimes looked at to see if a person is likely still contagious.  

The tests often look for at least 2 SARS-CoV-2-specific gene parts to be super sure. If both get amplified, the test is positive; if one gets amplified, the test is inconclusive; and if neither gets amplified, the test is negative (although there is always a chance of a “false negative” especially if the samples got degraded – remember how fragile RNA is!)⠀⠀ 

Of course, this is all assuming that the negative control (a sample of RNA that’s not from SARS-CoV-2) came back negative and the positive control (a sample of RNA that you know is from SARS-CoV-2) came back positive – if they didn’t, there’s something wrong with the test and the results are invalid. ⠀ 

RT-PCR is *really* sensitive – because each original RNA copy that’s there gets exponentially amplified. So a tiny amount of contamination can cause a negative sample to test positive (a so-called “false positive”). 

more on RT-PCR here: http://bit.ly/rtqpcroverview  

The RT-PCR tests are just one way to test for the virus – another way is with similar tests that do the DNA-copying using faster “isothermal amplification” methods: https://bit.ly/idnowrapidtests  

And another way is to look for viral proteins using “antigen tests”…

antigen tests

Firstly, Do NOT confuse this with an ANTIBODY TEST! Finding a viral antigen (viral “piece”) in someone tells you the virus is in that someone – now. It’s like catching a robber red-handed. But finding an anti-viral antibody in someone is more like finding a fingerprint the robber left on his way out – it tells you someone was infected in the past (or is currently recovering). Biochemically, antigen tests works similarly to an antibody test, BUT in REVERSE. They look for a viral protein, typically the viral N (nucleocapsid) protein. Here’s how…  

Antibody tests are used to detect little proteins called antibodies that your body makes to specifically bind to viral parts to help fight off infections and prevent re-infection. Antigen tests look for those viral parts, which are called antigens and are usually parts of viral proteins. So, these tests give you very different information. Antibody tests tell you if someone “has been” infected – in the past or currently (though antibodies don’t show up until later in the infection). Antigen tests tell you if someone is currently infected – they detect the virus itself which is only there when you’re actively infected, so these tests don’t tell you anything about past infections. Antigen tests are thus a form diagnostic test, along with tests like RT-PCR tests. 

Format-wise, they’re “lateral flow assays” – strips in a cassette similar to pregnancy tests, but instead of peeing on them, you “snot on them”. It’s not quite as simple of just blowing your nose (but wouldn’t it be awesome if in the future they had Kleenex that changed color in response to specific infections?…). Instead, how it works is that you stick a swab in your nose (one of those giant not-q-tips), swirl it around a bit to get a good sample, and then swirl that around in some “magic liquid” that has chemicals that break open the viral particles (lyse them).⠀ ⠀ 

The exact contents of the magic liquid are proprietary, but it contains detergents – these look similar to the lipids making up the membrane, so they’re able to wedge their way in between those lipids and break up the membrane, allowing the viral contents to spill out. (this is also why soaps and detergents (which are just synthetic soaps) are able to kill the virus: https://bit.ly/soapsanddetergents )⠀ 

Now that N’s out, the test needs to capture it and show it to us. ⠀ 

This capturing is actually done with anti-N antibodies. But these aren’t antibodies that come from the person being tested. Instead, they’re antibodies that have been made in a lab, attached to a colored or fluorescent thing and put onto that strip of paper in the cassette. ⠀ 

These tests use antibodies for the same reason your body does – they’re good at specifically binding viral pieces, helping the body do things like flag infected cells for destruction, call for backup, and keep watch after the virus has been conquered in case it tries to return (which is why antibody tests can detect past infection). ⠀ 

At the molecular level, antibodies (aka ImmunoGlobulins) are “just” little proteins which have generic adapter parts (constant regions) as well as unique parts (variable regions) that allow them to bind specifically to different viral parts (antigens). “Antigen” is just a shorter way of saying “that specific thing that the antibody binds to” and it typically is part of a viral protein. Terminology-wise, we typically specify the antigen after “anti-“ So, an anti-N antibody is an antibody whose antigen is the N protein. And these are the ones the test uses – in a couple different ways.⠀ 

Note: There are different layout strategies for doing these lateral flow tests & I don’t know exactly how each specific one’s organized inside the cassette since they keep those details inside the company as far as I can find. So I’m just going to describe one of the common schemes that is used. https://www.jacksonimmuno.com/technical/products/applications/elisa/lateral-flow/immunoassays-introduction

When you stick that  sample of spilled-out viral “guts” onto one end of the strip, the viral gut parts get a chance to interact with labeled beads or particles attached (conjugated) to anti-N antibodies. If there is N protein present, the anti-N antibodies will latch on. And then, thanks to capillary action, the mix gets wicked through the paper strip to the other end, similarly to how water spreads across a paper towel. (capillary action seems like magic but it’s really just because water’s super sticky but also likes to spread out, so it pulls itself through spaces in the paper’s fibers, dragging its friends with it).  ⠀ 

So, now you have your N protein bound to an anti-N antibody that’s bound to a fluorophore. All that’s left is to catch it. And we need to catch it separately from any antibody that is unbound, so we don’t confuse them. This is where the second set of antibodies comes in. A line of stuck-on anti-N antibodies is waiting to greet them when they reach that part of the strip. These bind to the (now-labeled) N protein – if it’s present. If the N protein isn’t present, the labeled antibody will keep flowing through to the wicking pad. So the labeled antibody will get stuck on the T line if N is present, but not if it isn’t. Now you just need to look to see if it’s there. 

Because, for a negative test, you’re looking an absence of a signal, you want to make sure that there’s not just no signal because the test is defective. This is where the control line comes in. The details vary by test but usually there’s a second labeled antibody present on that conjugate pad. It’s not going to stick to anything in the sample, but it will stick to antibodies on the second, control line (C) that are specific to it. So you should always see a line on the control line, regardless of whether the test is positive or negative.  

Antigen tests are less sensitive than PCR tests, but that’s not necessarily a problem if what you’re using them for is to detect whether a person is infectious. More here: http://bit.ly/reallyrapidtests 

more coronavirus resources (including details on how the other tests work) here: https://bit.ly/covid19bbresources

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