All around the world, people are facing the threat of the novel coronavirus, Covid-19, and the threat of misinformation and confusion, including about how the tests for detecting the causative virus, SARS-Cov-2, work. But I’ve been heartened to see that, around the world, scientists have been coming together to help clear things up – last week I put out a post about how these tests work and people seemed to find it helpful so, working with the IUBMB, we 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 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 and you’ll be able to find (and information about their translators) HERE
I know that my original version was pretty long – and for people who want a lot of details you can find it here: http://bit.ly/39UfWNc
But for those who just want the gist, here’s the “Cliff’s notes” version…
Most current tests for SARS-Cov-2 (the virus that causes the disease Covid19) are based on using a technique called PCR to look 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.
In PCR (Polymerase Chain Reaction), short pieces of DNA called primers are used 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
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
You can stop reading here, because that’s the gist. BUT, if you are interested in the amazing biochemistry that makes all this possible, keep reading… 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.
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, combined with non-optimal primers, may have contributed to problems with the original CDC test kits. Compounding the situation, until recently, other labs were restricted from producing their own tests for the virus, so it has taken a long time for testing to be conducted in the U.S. and there is still a great need to ramp it up.
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. But even if the PCR part is greatly sped up, RNA extraction is still more complicated and time-consuming
The RT-PCR tests are just one way to test for the virus – and it only detects 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 RT-PCR 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 the RT-PCR way can.
I never would have imagined that I would have a post translated into another language, let alone the dozen or so in the works. And I am truly and incredibly humbled and appreciative. These are hard times for all of us and it feels so gratifying to be able to do something helpful. I’m incredibly privileged to have had an amazing education and supportive family and colleagues, which have provided me with the knowledge and resources to contribute through post-making, but there are ways in which anyone can contribute, regardless of their background. 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…”