Finding a virus is a non-trivial scientific procedure. Equipment shortages and other challenges have limited who can be tested for COVID-19 in the U.S.A. Expanding the capacity for accurate, fast testing a serious challenge; we can’t solve it here, but hopefully you’ll walk away from this article with a bit more insight into the mechanics of COVID-19 testing, and some hope for the solutions that scientists are actively and swiftly developing.

Article by Rachel Jones, Little Shop of Physics Science Writer (rrjones@rams.colostate.edu)

If you have symptoms

If you have questions or concerns about non-emergency symptoms you may have, please call your primary healthcare provider or try a telehealth line. At the time of writing (April 2020), an interactive “self-checker” is available to people in the U.S.A. on the Centers for Disease Control and Prevention’s main coronavirus page. If you develop an “emergency warning sign” of COVID-19 (including, but not limited to, difficulty breathing or ongoing pressure in your chest), get medical care right away, and call ahead so that the providers can prepare. If you have a medical emergency while experiencing COVID-19 symptoms, call 911, and let the dispatcher know that you are sick.

The take-home message

How do you find a virus? They’re much too small to see even with the best light microscopes, and in the case of COVID-19, we can’t even count on just finding the people who are sick — evidence increasingly suggests that even people with no symptoms may be able to spread the coronavirus. This potential finding makes it particularly important that we do have a reliable way to figure out who’s carrying the virus. Currently, our best strategy for doing this is to look not for the coronavirus itself, but rather bits of genetic code that we think are unique to it.

To test a person for COVID-19, a healthcare worker swabs the back of their nose and throat to collect a sample. Laboratory workers then perform a procedure that separates the genetic material of any viruses present from the rest of the sample. Once this extraction is complete, workers get ready to perform a protocol that will make lots of copies of (“amplify”) certain coronavirus genes, but which will not amplify the genes of other viruses or of human cells. To the extracted genetic material, workers add molecules that will light up when a new copy of a coronavirus gene is made, enzymes that can copy genes, and the raw materials to make new DNA molecules. 

Workers then put these mixtures in a machine that provides the right conditions for copying and detects the flashes of light. After running for a while, the machine gives a readout that tells the worker how much light was made during each round of copying. The amount of light corresponds to the number of copies of viral genes. However, right now, we’re mainly concerned with figuring out whether the virus is present or not in a person (rather than figuring out the precise viral load), so the presence of light is a quick way to “see” the presence of the virus.

Clearly, the current testing procedure involves many steps and materials. Healthcare workers collecting samples need swabs to take samples with, as well as protective equipment such as face masks, gloves, and face shields. Labs need the chemicals that extract the genetic material, as well as the reagents and machines to amplify and detect the virus’ genetic material. And of course, at every step of the way, the people performing the testing need to be healthy.

We can help limit the strain on testing and healthcare workers by practicing social distancing, washing our hands, and self-isolating when sick, but scientists are also working on ways to make tests faster and more resource-efficient. One new test, recently authorized for emergency use by the FDA, uses a device that lots of testing centers may already have and can give results in 15 minutes or less. Testing for COVID-19 is very important, and currently a challenge in the U.S.A., but research is moving quickly, and we are getting better and better at finding the coronavirus.

The deep dive

Most virions have dimensions on the order of tens to hundreds of nanometers (e.g., [1 a, b, c]); your cells are hundreds of times wider. Virions are substantially smaller than the resolution limit of most light microscopes, so we can’t check for SARS-CoV-2 just by looking at samples in a typical lab. We can visualize virions with electron microscopes, as in Figure i, but these devices are highly specialized and far less accessible than light microscopes. Still, it is critical to determine who is infected with SARS-CoV-2, particularly given recent preliminary evidence that individuals with no COVID-19 symptoms may still be contagious [2] [3]. 

So, instead of looking for the virions themselves in samples from patients’ throats and noses, we look for several SARS-CoV-2 genetic signatures using a method called quantitative real-time reverse transcription polymerase chain reaction, henceforth real-time RT-PCR. (You may see this called “quantitative real-time PCR”, aka qPCR. The terms “qPCR” and “real-time PCR” both refer to the same technique; different scientists simply use different terminology.) There’s a whole lot of molecular genetics going on in that phrase, so let’s take a few words to understand it, working backward through the name.

This is a highly magnified, colorized image of SARS-CoV-2 virions; it was made using an electron microscope. The virions appear roughly round, and have many dim projections (which are spike proteins) around their circumferences.
Figure i: This is a highly magnified, colorized image of SARS-CoV-2 virions; it was made using an electron microscope. The virions appear roughly round, and have many dim projections (which are spike proteins) around their circumferences.

PCR: First, let’s talk about the fundamental basis of the test: polymerase chain reaction. PCR, a technique to make many copies of a particular gene or region of DNA from a small amount of “starter” genetic material, is one of the most significant advances in the field of molecular biology [4] [5]. To run a PCR, you first need to figure out a little bit about the gene you want to amplify so you can design primers. Primers are short snippets of DNA which bind to either side of the gene you want to amplify; they are what make the PCR specific to your gene of interest. An enzyme — DNA polymerase — needs these primers as a “starting block” to synthesize copies of your gene of interest.You’ll also need to add nucleotides the polymerase can use to build copies of the gene [4] [5]. Reagents for the CDC’s RT-PCR COVID-19 test kit are shown in Figure ii.

This is an image of materials provided in the CDC’s COVID-19 test. A small open box contains four very small vials of reagents secured in styrofoam. The lid of the box is visible in the background; it bears the CDC’s logo and reads, in part, “CDC 2019-nCoV real-time RT-PCR Panel… For research use only… Not for use in diagnostic procedures”.
Figure ii: This is an image of materials provided in the CDC’s COVID-19 test. A small open box contains four very small vials of reagents secured in styrofoam. The lid of the box is visible in the background; it bears the CDC’s logo and reads, in part, “CDC 2019-nCoV real-time RT-PCR Panel… For research use only… Not for use in diagnostic procedures”.

All these elements, including your sample, are combined and placed in a machine called a thermocycler [4]. The thermocycler, unsurprisingly, cycles temperature, precisely heating up and cooling down. The thermocycler first heats up to split apart the sample DNA double helix and give the enzyme access. It then cools down a bit to allow the primers to bind, and then a bit more to allow the enzyme to do its work and copy your gene of interest. These steps are repeated many times; each time, there are more copies of your gene of interest to provide templates for copying, so the number of copies increases exponentially (hence “chain reaction”) [4] [5].

RT: This basic procedure is commonly used in molecular biology studies around the world, but it can also be modified to suit unique applications and answer particular questions. Standard PCR can tell you if a given fragment of DNA is present, but it can’t give a great deal of information about how much DNA you started with, and it doesn’t work with RNA. RNA is the molecule into which snippets of the genomic DNA code are transcribed. Different types of RNAs serve different functions, but we’ll talk here about messenger RNA (mRNA), which is translated into proteins. The polymerase used in PCR can only copy DNA, so if you want to study RNA using PCR, you’ll need an additional step before the thermocycler: treatment with the enzyme reverse transcriptase [4]. Figure iii is an illustration of the HIV reverse transcriptase. In “normal” transcription, DNA is coded to RNA, but reverse transcriptase catalyzes the opposite reaction — reverse transcription (hence “RT”-PCR), the coding of RNA to DNA.

This is a representation of the HIV reverse transcriptase. The coils and arrows represent different basic protein structures (specifically, alpha helices and beta sheets). The figure also includes several ball-and-stick molecule models, which indicate the active sites (where the reaction catalyzed by the enzyme happens).
Figure iii: This is a representation of the HIV reverse transcriptase. The coils and arrows represent different basic protein structures (specifically, alpha helices and beta sheets). The figure also includes several ball-and-stick molecule models, which indicate the active sites (where the reaction catalyzed by the enzyme happens).

An important question: Why do we need reverse transcriptase to find the genes of SARS-CoV-2? This has to do with the basic biology of the virus. SARS-CoV-2 is a coronavirus, and like other coronaviruses, is an RNA virus, aka retrovirus [6] [7]. Retroviruses are rather unusual in that their genomes are written entirely in RNA, not DNA [5]. A typical retrovirus virion consists of a number of RNA molecules surrounded by a protein capsid and a lipid bilayer. Each RNA molecule in the virion is paired with a molecule of reverse transcriptase. When the lipid bilayer of the virion fuses with the lipid bilayer of a host cell, the viral RNAs and enzymes are released into the host cell, wherein viral reverse transcriptase codes the viral RNA to DNA. The viral DNA then becomes part of the host cell’s genome. Since the viral genes contain bits of code that instruct the host cell to make many, many copies of them, the host cell essentially becomes a factory for new retroviruses, which can leave the host cell and infect neighboring cells [5].

real-time: So, now that we understand the acronym portions of “real-time RT-PCR”, we come to the phrase “real-time”. This moniker refers to the technique of capturing the results of the PCR as genes are being amplified, rather than using gel electrophoresis to examine the PCR products after the reaction is done. Seeing PCR results in real-time generally requires adding fluorescent dyes to the PCR reaction, and running the reaction in a machine that can detect and precisely measure flashes of light from the dyes [4]. Although there are a number of protocol available for real-time PCR, we will focus on one particular widespread approach: the use of TaqMan probes.

Like primers (the short DNA “starting blocks” for DNA polymerase), TaqMan probes are specific to your gene of interest, but unlike primers, TaqMan probes contain a molecule that can fluoresce [4]. This molecule is called a “reporter”. Now, what’s really clever about TaqMan probes is the pairing of this reporter with a “quencher”, which, when close the reporter, prevents detection of light emitted by the reporter. During a PCR cycle, a TaqMan probe can bind to your gene of interest. However, due to the presence of the quencher, light from the probe won’t be detected until DNA polymerase pops the reporter off the probe as the enzyme reads and copies your gene of interest. As the reporter moves away from the quencher, the PCR machine is able to detect the reporter’s fluorescence, which fluorescence directly implies the presence of your gene of interest (since the probes are specific to that gene) [4]. Figure iv is a diagrammatic representation of the use of TaqMan probes in real-time PCR.

This diagram illustrates the use of TaqMan probes in real-time PCR. Initially, the probe, containing a reporter (“fluorophore”) and a quencher, is bound to a strand of DNA. As the strand of DNA is copied, the reporter breaks away from the quencher, and light from the reporter becomes detectable. Eventually, the whole probe falls off the DNA as copying proceeds.
Figure iv: This diagram illustrates the use of TaqMan probes in real-time PCR. Initially, the probe, containing a reporter (“fluorophore”) and a quencher, is bound to a strand of DNA. As the strand of DNA is copied, the reporter breaks away from the quencher, and light from the reporter becomes detectable. Eventually, the whole probe falls off the DNA as copying proceeds.

final thoughts: At this point, we have truly put together a picture of the current “gold standard” of COVID-19 testing. The CDC’s own test is a real-time RT-PCR assay that uses TaqMan probes to detect two different SARS-CoV-2 genes [8]. However; at this point it is also likely clear that this test requires many reagents, advanced laboratory equipment, and specialized technical knowledge. In practice, it is currently not possible to test everyone in the U.S.A. who would like a test.The CDC emphasizes that not everyone needs a test, and the Colorado Department of Public Health and Environment has developed a set of categories to allow preferential testing of those for whom a positive result would have the greatest medical or public health impact.

Research is moving quickly to expand our currently limited testing capacity. The FDA currently (April 2020) lists many Emergency Use Authorizations (EUAs) for COVID-19 tests. One such authorization is for a test based on isothermal nucleic acid amplification, a relatively new technique but one that uses devices many labs may already have, and which gives results in less than 15 minutes [9]. Some early research suggests strategies that may allow for more widespread testing by easing the need for particular reagents that are used to extract viral RNA from samples [10] [11]. Other preliminary results open the possibility of using technology that could test many more samples simultaneously than is currently possible [12]. However, it is critical to remember that this research is still precursory, and it is necessary to ensure that application of these methods will still provide accurate testing results in a clinical setting. While there is hope for faster, wider-spread testing, we still must take care of ourselves and others in our communities by practicing social distancing, washing our hands, and carefully monitoring ourselves for symptoms.

SARS-CoV-2 may be using reverse transcriptase to co-opt our cells’ machinery as it makes us sick, but we’re using its own trick against it every time we test for COVID-19. And we keep getting better at doing so.

References

[1] Milo R, Jorgensen P, Moran U, Weber G, Springer M. 2010. BioNumbers — the database of key numbers in molecular and cell biology. Nucleic Acids Research 38: D750-D753. 

[2] Rothe C, Schunk M, Sothmann P, Bretzel G, Froeschl G, Wallrauch C, Zimmer T, Thiel V, Janke C, Guggemos W, Seilmaier M, Drosten C, Vollmar P, Zwirglmaier K, Zange S, Wölfel R, Hoelscher M. 2020. New England Journal of Medicine 382: 970-971. Available from https://doi.org/10.1056/NEJMc2001468

[3] Wei WE, Zongbin L, Chiew CJ, Yong SE, Toh MP, Lee VJ. 2020. Presymptomatic transmission of SARS-CoV-2 — Singapore, January 23-March 16, 2020. Morbidity and Mortality Weekly Report 69: 411-415. Available from http://dx.doi.org/10.15585/mmwr.mm6914e1

[4] Klug WS, Cummings MR, Spencer CA, Palladino MA. Concepts of Genetics (11e). Boston (MA): Pearson; 2015. 752 p.

[5] Hardin J, Bertoni G, Kleinsmith LJ. Becker’s World of the Cell (8e). Boston (MA): Pearson; 2015. 793 p.

[6] Zhou P, Yang X-L, Wang X-G, Hu B, Zhang L, Zhang W, Si H-R, Zhu Y, Li B, Huang C-L, Chen H-D, Chen J, Luo Y, Guo H, Jiang R-D, Liu M-Q, Chen Y, Shen X-R, Wang X, Zheng X-S, Zhao K, Chen Q-J, Deng F, Liu L-L, Yan B, Zhan F-X, Wang Y-Y, Xiao G-F, Shi Z-L. 2020. Nature 579: 270-273.

[7] Sah R, Rodriguez-Morales AJ, Jha R, Chu DKW, Gu H, Peiris M, Bastola A, Lal BK, Ojha HC, Rabaan AA, Zambrano LI, Costello A, Morita K, Pandey BD, Poon LLM. 2020. Microbiology Resource Announcements 9: e00169-20. Available from https://doi.org/10.1128/MRA.00169-20

[8] Centers for Disease Control and Prevention Division of Viral Diseases (USA). CDC 2019-novel coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel for emergency use only instructions for use [Internet]. Atlanta (GA): Centers for Disease Control and Prevention (USA); 2020 March 30 [cited 2020 April 11]. Available from https://www.fda.gov/media/134922/download

[9] Abbott. ID NOWTM COVID-19 [Internet; page and linked Product Insert]. 2020 [cited 2020 April 11]. Available from https://www.alere.com/en/home/product-details/id-now-covid-19.html

[10] Beltrán-Pavez C, Márquez CL, Muñoz G, Valiente-Echeverría F, Gaggero A, Soto-Rifo R, Barriga GP. SARS-CoV-2 detection from nasopharyngeal swab samples without RNA extraction. Preprint posted to bioRxiv 30 March 2020. Available from https://doi.org/10.1101/2020.03.28.013508

[11] Bruce EA, Tighe S, Hoffman JJ, Laaguiby P, Gerrard DL, Diehl SA, Leonard DGB, Huston CD, Kirkpatrick BD, Crothers JW, Dragon J, Botten J. RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swab using Qiagen RNeasy kits or directly via omission of an RNA extraction step. Preprint posted to bioRxiv 6 April 2020. Available from https://doi.org/10.1101/2020.03.20.001008

[12] Nelson AC, Auch B, Schomaker M, Gohl DM, Grady P, Johnson D, Kincaid R, Karnuth KE, Daniel J, Fiege JK, Fay EJ, Bold T, Langlois RA, Beckman KB, Yohe S. Analytical validation of a COVID-19 qRT-PCR detection assay using a 384-well format and three extraction methods. Preprint posted to bioRxiv 5 April 2020. Available from https://doi.org/10.1101/2020.04.02.022186

Image credits

[i] “Electron micrograph of SARS-CoV-2 virions with visible coronae” by the National Institute of Allergy and Infectious Diseases (NIAID). Used under CC BY 2.0. Available from https://en.wikipedia.org/wiki/Severe_acute_respiratory_syndrome_coronavirus_2#/media/File:Novel_Coronavirus_SARS-CoV-2.jpg

[ii] Image of SARS-CoV-2 test kits provided by the U.S.A. Centers for Disease Control and Prevention (public domain).

[iii] “HIV-1 Reverse Transcriptase with Active Sites” by MLGProGamer123 – own work. Used under CC BY-SA 4.0. Available from https://en.wikipedia.org/wiki/Reverse_transcriptase#/media/File:HIV-1_Reverse_Transcriptase_with_Active_Sites.png

[iv] “Taqman probes” by User:Braindamaged – own work. Public domain. Available from https://en.wikipedia.org/wiki/Reverse_transcription_polymerase_chain_reaction#/media/File:Taqman.png / Polarity of DNA strands corrected to indicate 5’ to 3’ polymerization