TESTS! Oh, the good ole days when people dreaded taking tests… These days, everyone is *wanting* to take tests – to see if they have or had Covid-19, the disease caused by the novel coronavirus, SARS-Cov-2. There are a lot of different variations of the tests, but they fall into 2 main types: diagnostic tests which look for active (& thus contagious) disease and serological/antibody tests, which look for evidence of past disease. Here’s an overview of how they work, and what you need to watch out for.
Let’s start with the diagnostic tests – the ones for seeing if you currently have the disease. Most current tests for SARS-Cov-2 are based on looking for genetic information specific to that virus, such as stretches of the genes it has for making proteins that it needs but we don’t have. There’s not much of this viral info, so scientists have to make lots of copies of it and use sensitive methods to detect it.⠀
The traditional tests do the copy-making using a technique called PCR (Polymerase Chain Reaction), which involves cycles of up-down-temperature changing. That process can a while (couple hours usually), so some “rapid tests” are using alternative copying mechanisms that are carried out at a single temperature. There are some differences, but these “isothermal amplification” techniques, as well as PCR (Polymerase Chain Reaction) use short pieces of DNA (called primers in the case of PCR) to specify a region of the viral genome to copy. Fluorescent probes bind to the copies and let off light which allows scientists to see copies as they get made – if they get made that is. You can’t make copies of something that isn’t there, so if scientists see fluorescence above a threshold set for background “noise,” a sample is considered positive for the virus – but if the fluorescence stays below the threshold, in the noise region, the sample is considered negative (although tests usually check for at least 2 targets to be really sure).
One “slight inconvenience” when it comes to SARS-Cov-2 is that it is a single-stranded RNA virus – it stores and transmits its genetic blueprint (genome) in the form of a single strand of RNA instead of double-stranded DNA like we have. But PCR works by making copies of DNA. So, after scientists extract the viral RNA from patient samples they convert it to DNA form in a process called reverse transcription before doing the copying. Thankfully, this is fairly easy, and it’s the extraction part that is the biggest time (and “needed things”) hold-up. Speaking of which, if you are a scientist with extraction kits to spare check out this page on the RNA society’s website to see how you can help: http://bit.ly/2IMCSSD One of the reasons some of the “rapid tests” (like Abbott’s ID NOW) are rapid is that they don’t require extraction – instead the machine does it all (but one sample at a time and with poor reliability… https://bit.ly/3bsCTI1 )
So the basic premise of the test is – doctor collects patient sample (often taken by swabbing the nose and/or throat) and sends it to a lab and then the lab scientists:
- extract the RNA
- reverse transcribe that RNA into DNA form in preparation for copying
- copy it and copy it and copy it… and detect the copies as they’re made
At its heart it comes down to the chemical makeup of these 2 main forms in which genetic information can be stored: DNA and RNA, collectively referred to as “nucleic acids.”
These can sound like really abstract concepts, but it’s not like “string theory” where who knows what the heck’s going on… instead it’s *strand* theory – because DNA and RNA are just physical strands of chemical “building blocks” or “letters” called nucleotides – ribonucleotides in RNA and deoxyribonucleotides in DNA. Nucleotides have a generic sugar-phosphate part that allows them to link up to form strands and one of 4 unique “bases” that stick off the strands and allow for complementary base pairing between strands. RNA and DNA both have the bases C, G, and A. They differ in the 4th base – DNA has “T” and RNA has “U” – but U and T are really similar and act the same when it comes to allowing for specific base pairing between strands – the chemical structure of the base “A” matches up with the structure of “T” (in DNA) or “U” (in RNA and the structure of “G” makes it like to stick to the base “C.” So you can have complementary base pairing between DNA and DNA or DNA and RNA or RNA and RNA, allowing for the 2 types of nucleic acids to stick together and to serve as templates for making one another.
For example, thanks to this specific base to base pairing ability, a strand of DNA can serve as a template to be used to copy a complementary strand of DNA which then can serve as a template for copying the original strand. Each strand that gets made can serve as a template, so you can do this over and over to exponentially get lots and lots of copies. This is the basic premise PCR: we can stick DNA we want copied into a little test tube and use short pieces of DNA called primers that are complementary to where we want to copying to start and stop (these primers are designed to bookend the copied region (amplicon).
It’s important that the primers are specific to the thing we want copied – they need to match a sequence that isn’t found anywhere else. In the case of the SARS-Cov-2 tests, this means a sequence that is in the genome of SARS-Cov-2, but not in our own genome or that of other potential disease-causers. Some of the currently available tests (including the CDC test) target regions of the N gene (the instructions for making the Nucleocapsid protein that forms a protective coat around the RNA) while others target the E gene (for an Envelope protein that gets embedded in the viral membrane) and/or the RdRP gene (the gene for making the RNA-dependent RNA polymerase the viral uses to make RNA copies of its RNA genome). It doesn’t really matter where they target, as long as that target is specific to SARS-Cov-2.
No matter where you copy, however, you need a way to detect the copies. In real time PCR, this is done using fluorescently-labeled DNA probes that bind to the copied region of each strand that gets made. The fluorescent part gets “freed” during the next copy cycle, allowing you to see the copies as they’re made. If you plot the fluorescence versus the number of amplification cycles, you get a curve you can use to see how many cycles it takes to cross the background threshold (if it ever does). This value is called the Ct value.
CRISPR-based methods are also being developed for a detection strategy once viral genetic info is copied (using PCR or an isothermal amplification technique). CRISPR is that gene editing tool from bacteria you might have heard of. You can program a “Cas” protein with specific RNA sequences and the Cas will go cut that sequence. For gene editing purposes, you want to make sure that Cas only cuts exactly where you want it too and then stop. But there are some versions of the Cas protein that, once making that specific cut, will cut up any single-stranded DNA that’s nearby. So if there are probes nearby, it’ll cut them, which, depending on the test’s design will either give off light, similarly to above, or allow the cut pieces to be separated and detected in a paper strip format. https://tcrn.ch/2VVD3Ru
There’s been this huge rush to develop newer, faster, tests because the US is way behind where we need to be in terms of testing. RT-PCR is a very sensitive technique, which is good because it allows for the detection of very small amounts of viral RNA but it also means that small amounts of contaminating RNA can cause “false positives” which caused problems with the original CDC test kits. Those kits included primer/probe sets to look for 3 different regions of the N gene, and they got some viral RNA into one of them. If you want to learn more about that, you can read more in this past post https://bit.ly/3cwB2Sy
But, bottom line is that it set the US back in terms of diagnostic testing capacity especially since, in the beginning, the CDC wasn’t letting other labs develop their own tests. But, now there are several different companies making PCR tests. And there are also efforts being made to semi-automate the process so that more tests can be done more quickly – the CDC protocol uses 96-well PCR plates (about the size of a big iPhone, with the wells about hole-punch-sized) that scientists have to manually pipet into. So, even though the PCR part only takes a few hours or less, the set up takes a while. Automated and semi-automated methods help speed this up but require more expensive equipment. And different companies have different equipment and different chemical mixes that go with their tests so there’s a lot of having to coordinate and get supplies, etc. But even if the PCR part is greatly sped up, RNA extraction is still more complicated and time-consuming⠀
So, methods like the rapid ID NOW test sound great because you don’t have to do that manual RNA extraction, BUT they give a lot of false negatives – so they tell people that they don’t have the disease even though that person DOES have the disease. This can lead to them not quarantining adequately, etc. because they’re told they’re good (or at least that they don’t have Covid-19, they still probably have something so should be staying away from people anyways…) More here: https://bit.ly/3bsCTI1
Another holdup point you might have heard about is the nasal swabs – those giant q-tip things that doctors stick super far up your nose to get a sample. Shortages of these has caused delays, but shorter swabs are now approved for use in some cases (like in the “at-home” kits, currently prioritized for first-responders, where you swab your own nose and mail back the swab). These shorter swabs take sample from closer to your nostril, which I’m assuming is a lot less painful, but it’s not yet clear if you alway get a good enough sample (you need it to have enough viral particles to detect). There are also companies looking to market spit-based tests (it’s harder because spit contains a lot of proteins that degrade the sample).
All these diagnostic tests only detect the virus it when people are still acutely infected, and the virus is still making all that RNA to make all the proteins it needs to make more of itself and infect more cells. Once the virus is “conquered” by a person’s immune system, that viral RNA isn’t there anymore; however, evidence of the proteins made from it is – the immune response that allowed the body to fight off the virus involved making little proteins called antibodies that recognize specific pieces of the viral proteins as “foreign” and trigger an immune response.
Some of these antibodies stick around after the infection’s over to “keep watch,” do tests that look for antibodies can see if someone previously had the virus, even after they’ve recovered, and this can be used to trace cases back to see the line of transmission even if the transmitters are no longer symptomatic and don’t have the RNA that the PCR or isothermal amplification tests could detect.
The antibody tests are quicker and they’re typically done on blood samples, but a downside with them is that, since they come from the immune response finally gaining some ground on the virus, they can’t detect the virus as early in an infection, while PCR or isothermal amplification ways can.
There’s been a LOT of hope put on these tests – especially hope that they can tell that people are immune to the disease and don’t have to worry. BUT BUT BUT there is no test that can guarantee against future infection from Covid-19. Some of the more sophisticated tests can look to how strong someone’s antibodies against it are and give a little more comfort, though no guarantee – and no knowing how long those antibodies will stick around. The less sophisticated “lateral flow” rapid tests that are like pregnancy tests that you finger-prick-bleed on don’t tell you about the strength of the antibodies. And they might not tell you ANYTHING at all. Because the FDA has been really lax about regulating these tests and there are A LOT of bad ones on the market. And, even the best antibody tests have some rate of false positives and false negatives – even if it’s a really low rate, if you test lots and lots of people like “serological prevalence” studies do to try to get a better sense of how widespread the disease was, you’re going to get lots of wrong results. https://bit.ly/rapidantibodytests
It’s been a month and a half since I posted my original (long-version) explanation of Covid-19 testing http://bit.ly/coronavirustesting
Since then, with the IUBMB, I put out a call for volunteers to translate the explanatory figures I’d made into additional languages – and got an outpouring of offers from around the world – from Africa to Europe and South America to Asia – Switzerland to Greece to Argentina and China. I’m currently still working on formatting them all, but will be adding them to my blog as I do. We’re up to 30!!!!! and you can find them (and information about their translators) here: https://bit.ly/covid19bbresources ⠀
I want to thank all of the translators. And I want to thank all of the people doing whatever they can to help others during this hard times. This can be as simple as calling to check in on a neighbor, delivering supplies, or even just staying home!
This last one is part of what’s referred to as social distancing – avoiding gathering in groups and, if you must, maintaining a 6-foot distance from those around you. As someone who LOVES lab work, it’s definitely gonna be a challenge to work from home for a bit, but I felt it was the right call for myself – since I have the privilege of staying home since I’m a “non-essential” worker, I want to do so in order to protect the people who have to go to work – from the janitors to the store clerks to, of course, the doctors and nurses at the front lines. Staying home may sound “boring” but it really can save lives – by practicing social distancing we can “flatten the curve” of infections over time so that we don’t overwhelm the health care system.
Social distancing can feel isolating, so be sure to use all those internet-ty tools – and even just your good ole’ phone – to connect with friends and family. And know that, even if I don’t know you personally, I am thinking of you – each and every one of you – and wishing you nothing but the best.
Now, more than ever, as we face an international (and biochemistry-related) crisis, I am incredibly grateful to be able to serve as Student Ambassador for the International Union of Biochemistry and Molecular Biology (IUBMB) that has helped me recruit translators and share the translated versions around the world. This post was just one in my series of weekly “Bri*fings from the Bench” which, for a while, will have to be “Bri*fings from the Bedroom…”
more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0