How it works: coronavirus testing
The Centers for Disease Control (CDC) encountered difficulties in rolling out the first diagnostic test for the novel coronavirus. In order to understand the problem, it’s important to understand how the test works at a molecular level.
SARS-CoV-2 took the world by surprise in late 2019 when a mysterious pneumonia began circulating through Wuhan, China. Scientists moved swiftly to identify the underlying pathogen as a novel virus from the family Coronaviridae, and its large RNA genome was quickly sequenced. Armed with genetic information, researchers were able to develop a simple test, called polymerase chain reaction or PCR, to determine whether a patient carried the virus. Some countries like South Korea deployed this type of test widely to identify the infected and isolate them, keeping the spread of the virus under control. Other countries like the United States faced significant challenges in launching diagnostic testing, which has raised questions about how the assay works, where we went wrong and what other diagnostic tests are in the pipeline.
How does the diagnostic PCR test work?
Cells use an enzyme called DNA polymerase to generate a second copy of their genome before they divide into two daughter cells. PCR takes advantage of the same enzyme to make many copies of a specific sequence of DNA to the point where it can be easily detected. But SARS-CoV-2 is an RNA virus, so how could we use PCR to detect it? The answer is that there is another enzyme called reverse transcriptase (RT) that can make a copy of DNA (complementary DNA or cDNA) from an RNA template. So step 1 in the PCR test is to use RT to make a cDNA copy of the viral RNA. With the new cDNA template, you’re now ready to do PCR amplification. A few components are needed for this step: DNA polymerase to make new copies, a short piece of DNA that is specific to the virus so the enzyme knows what to amplify (primer), the building blocks of DNA (nucleotides) and an extra piece of DNA called a probe. By changing the temperature of the reaction, we can control the activity of the DNA and the enzyme. At body temperature, DNA has two strands that form a double-helix, but at high temperatures these strands melt and separate. If we then lower the temperature, the DNA strands stick back together, but some of them will stick to the primers instead of to each other. This gives DNA polymerase the signal to attach to the DNA and start chugging along, making a copy using the nucleotide building blocks in the solution.
In an ideal situation, if we started with one copy of the viral cDNA, after the first cycle we have two. The temperature cycling is then repeated forty times to produce up to 2^40 (1 trillion!) copies of viral cDNA. But how do we know it’s working? That’s where the probe comes in. It’s a piece of DNA that sticks to the new viral cDNA copies, and it has a molecule on the end of it called a fluorophore that becomes fluorescent (lights up) when it runs into DNA polymerase. So scientists can tell how much viral RNA a sample has based how much light it produces.
Why did the PCR test have problems in the US?
The most common problem in PCR is bad primers. The enzyme, the detection system, and the temperatures are all pretty well figured out and they don’t vary much between assays, but primers must be designed for each new target. These short pieces of DNA sometimes stick to themselves more than to the template (false negative) or they stick to similar pieces of DNA in the sample other than their intended target (false positive). Therefore it’s crucial when designing primers to pick a stretch of SARS-CoV-2 RNA that is unique to the virus and doesn’t overlap with similar coronaviruses, for example. However, this doesn’t really explain the issue with the CDC’s test: they were getting a fluorescent signal even in distilled water, with no cDNA template added. PCR is extremely sensitive: it can amplify even trace amounts of target DNA to the point of detection. So if any of the reagents in the kit (enzyme, primers, nucleotides, water, controls) are contaminated with target DNA, all samples will test positive. So it’s likely that when the CDC started putting test kits together, one or more of the reagents was contaminated by their positive controls (which contains SARS-CoV cDNA). That means the test was basically unusable, and it really set back the public health labs that were relying on it to get out in front of the outbreak early. Fortunately, the FDA has now granted Emergency Use Authorization (EUA) for over 30 RNA-based diagnostic tests and testing has since ramped up.
Is there another way to test for SARS-CoV-2 RNA?
The FDA recently approved a diagnostic test developed by Abbott Laboratories called ID NOW COVID-19. This method takes advantage of a strategy called isothermal amplification, which uses many of the same principles as PCR but does not require temperature cycling. Instead, this test uses a DNA polymerase that can separate DNA strands on its own, without the need for high temperatures. The Abbott Laboratories test is much faster than traditional PCR, taking only 5–30 minutes instead of two hours. And importantly, the new test can be run right in the doctor’s office, unlike the PCR test which must be done in a lab. But the ID NOW COVID-19 machine can only process one sample at a time, compared to a PCR machine that can do 384 tests simultaneously. So ID NOW will probably be more useful for private doctor’s offices and urgent care centers, while hospitals will most likely continue to use PCR to do large batches of tests.
Are there other ways to detect the virus?
Instead, of reinventing the wheel, it’s much more efficient to take advantage of molecules that already exist in biology to do the work of detecting the virus. The human immune system is very good at identifying pathogens, and SARS-CoV-2 is no exception. The novel coronavirus primarily infects cells along the respiratory tract, which is patrolled by immune cells called macrophages. A macrophage will start engulfing viral particles to break them down, and some of these cells can display pieces of the virus on their cell membrane to alert other immune cells of the attack. This activates a more specific response to the virus from T cells, which kill infected cells, and B cells, which produce proteins called antibodies specific to the virus. These antibodies stick to viral particles to inactivate and flag them so other immune cells can easily find and destroy them. Early in an infection, B cells produce a large antibody called Immunoglobulin M (IgM), and later they produce smaller a smaller antibody called IgG. So if we can detect IgM antibodies specific to SARS-CoV-2 in a patient’s blood sample, it’s likely that they have a current or recent infection, while SARS-CoV-2-specific IgG molecules would suggest a recent or previous infection.
The FDA recently approved a test by Cellex to detect these two types of antibodies using a lateral flow immunoassay, which just means they run plasma (blood without cells) through a membrane with SARS-CoV-2 proteins on it and see if anything sticks. It is similar in principle to a pregnancy test: a colored line develops if the sample is positive.
As plasma moves across the membrane, it first encounters SARS-CoV-2 proteins which are labeled red. If the sample contains antibodies against the virus, they will pick up the red-labeled proteins. Further along, the membrane has antibodies that recognize human IgM and human IgG (yes, antibodies that recognize other antibodies). If the sample has IgM antibodies against the virus, they’ll stick to the first line (M) and turn it red. If the sample has IgG antibodies against the virus, they’ll stick to the second line (G). The last line is a control © to make sure the fluid is moving through the membrane properly. If the patient is negative for SARS-CoV-2 IgM and IgG, it’s unlikely that they have been exposed to the virus. If they have either or both IgM and IgG antibodies they have almost certainly been exposed at some point. This test will be really useful for healthcare workers who suspect they’ve been infected, especially if they feel better and want to go back to work. Eventually, the antibody test will be critical for determining who in the general population has immunity.
More information
CDC test issues: https://www.businessinsider.com/early-coronavirus-cdc-tests-distinguish-covid-water-2020-3
ID NOW COVID-19 test: https://www.alere.com/en/home/product-details/id-now-covid-19.html
Isothermal amplification: https://www.neb.com/applications/dna-amplification-pcr-and-qpcr/isothermal-amplification
Cellex antibody test: https://www.fda.gov/media/136625/download
Nicole Aiello is a postdoctoral fellow who studies the role of stem cells in normal mammary gland development and breast cancer. She earned her PhD in Cell and Molecular Biology from the University of Pennsylvania.
