SARS-CoV-2 Testing Methods

Published on 4 Dec 2020


Since the declaration of a pandemic in March 2020, there have been strong efforts by laboratories and industry to make diagnostic tests available to identify and control COVID-19, which is caused by the virus known as SARS-CoV-2. The first type of test to hit the market were molecular assays, most commonly real-time PCR tests. These have continued to be the gold-standard method for diagnosis of COVID-19. However, there have been challenges often related to the global shortage of swabs to perform sample collection from patients, as well as the transport media for those swabs due to the unprecedented demand and the need for massive scale up in a very short time.

In the months after molecular diagnostics were available, other technologies to test for COVID-19 have become available. Rapid development of lateral flow immunoassays also took place, which could be used for point-of-care detection of antibodies in patients with presumed exposure to SARS-CoV-2. Subsequently, laboratory-based serology tests have been implemented in a number of clinical laboratories for detection of SARS-CoV-2 antibodies. Most recently, we’ve seen a significant amount of interest in rapid, point-of-care antigen tests, of which there are now multiple commercial options that have been authorized for use. Each of these testing options comes with specific advantages and limitations.

Objectives include:

  • Ensure the participants know what types of diagnostic technologies have been developed for detection of SARS-CoV-2.
  • Identify when each type of test should be used during the course of the disease and on which type of individuals they are most appropriate.
  • Understand the strengths and limitations of each type of test when it comes to detecting SARS-CoV-2 in patients.



Matthew Binnicker, PhD

Director of Clinical Virology Professor of Laboratory Medicine and Pathology, Mayo Clinic


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Webinar Transcript:

“Hi, my name is Dr. Matt Binnicker and I am the Director of Clinical Virology and a Professor of Laboratory Medicine and Pathology at the Mayo Clinic in Rochester, Minnesota.

Since the declaration of a pandemic in March of 2020, there has been a significant amount of effort by laboratories and industry to make diagnostics available to diagnose and manage COVID-19, which is caused by the virus known as SARS-CoV-2.

Molecular techniques were the first to become available and remain as the gold standard in this pandemic. However, there have been challenges often related to the global shortage of swabs to perform sample collection from patients, as well as the transport media for those swabs due to the unprecedented demand and the need for massive scale up in a very short period of time. 

In the months after molecular diagnostics became available, other technologies to test for COVID-19 have become available, each with specific advantages and limitations.

Let’s take some time to understand what types of diagnostic technologies have been developed, when they should be used during the course of the disease and on which type of individuals they are most appropriate.

Let’s start by summarizing the type of tests that are currently available for diagnosing COVID-19. One of the silver linings of the COVID-19 pandemic has been the speed and scale of diagnostic innovation. There has been so much activity in this space, and it’s good to review what types of technologies have been developed and what tests are currently available. The first type of test to hit the market were molecular assays, most commonly real-time PCR tests. These have continued to be the gold-standard method for diagnosis of COVID-19. In addition to molecular tests, there was also rapid development of lateral flow immunoassays, which could be used for point-of-care detection of antibodies in patients with presumed exposure to SARS-CoV-2. Subsequently, lab-based serology tests have been implemented in a number of clinical laboratories for detection of SARS-CoV-2 antibodies. And most recently, we’ve seen a significant amount of interest in rapid, point-of-care antigen tests, of which there are now multiple commercial options that have been authorized for use.

I know it’s difficult to keep track of the rapidly changing landscape of COVID-19 diagnostics with so many test types and options. There is an ever growing number of testing options and it’s difficult to keep track of how these different methods work, when they should be used, and for what type of patients they’re most appropriate. So I’d like to break down this information, starting with molecular tests. As I mentioned earlier, molecular tests, like real-time PCR, were the first type of method broadly available for the diagnosis of COVID-19. This category of test works by identifying whether the virus’ genetic material, or the RNA, is present in a patient’s clinical sample. Let’s walk through the common steps involved in a molecular test for COVID-19.

First, a sample is collected from a patient who is suspected of being infected with SARS-CoV-2. This sample is most commonly a swab of the patient’s nose or nasopharynx, which is the back part of the nasal passage where it meets the upper part of the throat. The swab is then placed in a tube containing a fluid, called transport media that helps to stabilize that sample prior to testing. Once the sample arrives at a lab, the most common next step is to extract, or purify out, the virus’ genetic material – that RNA – from everything else that is present in the specimen. After the viral RNA has been purified, it’s ready to be tested. The most common molecular approach is called PCR, which stands for polymerase chain reaction. PCR targets a very small part of the virus’ RNA, and over the course of minutes to hours, amplifies – or copies – that single region of the virus’ genome into potentially tens-of-millions of copies. Enough for the PCR instrument to detect – or ‘see’ that – because a fluorescent signal is produced each time the virus’ gene region is replicated.

Even though the general principle of amplifying, or making copies of a part of the viral genome is shared among most COVID-19 molecular tests, there are some important differences to highlight. For example, some tests require a separate “extraction” step, where the viral RNA is purified before the PCR test is performed. These tests usually take a bit longer to perform, typically in the range of 4-8 hours after the sample arrives at the lab. However, they can usually perform a larger number of tests each day, so they’re often well suited for labs that are performing hundreds or thousands of tests on a daily basis. (6min)

Some other molecular assays incorporate that RNA extraction step into the test process. These ‘sample-to-answer’ systems are faster, and can provide results in less than one hour in some situations. These types of systems are useful when rapid results are needed for patient management decisions.

Now that we’ve reviewed how molecular tests work its important to review the factors that are often considered when a physician orders a molecular test for COVID-19. As we discussed earlier, these types of tests are designed to tell us whether the virus’ RNA is present in a patient’s respiratory tract or not. So they are best suited for diagnosing someone with active disease. We’ve learned a lot about SARS-CoV-2 over the past 9 months, and one of the things we’ve discovered is that this virus is present at the highest amounts around the time someone develops symptoms. This is usually about 5 days after they’re exposed to the virus. So we recommend that a molecular test, like PCR, be ordered as soon as possible after a patient develops symptoms, or if there was a high risk exposure and the person is asymptomatic, waiting until about 5 to 7 days after the exposure to test by PCR, when there is the highest likelihood that the virus will be present in the upper respiratory tract.

I should also point out that we no longer recommend that PCR be used as a ‘test of cure’, or in other words, to determine whether someone still is infectious. This is because PCR can continue to be positive for weeks or even months in patients who have been infected with SARS-CoV-2, even after they have recovered from their illness. Instead, the United States Centers for Disease Control and Prevention, or CDC, recommends that we us a symptom- or time-based strategy to determine when an individual can be released from isolation or quarantine.

So to reemphasize, molecular tests  can tell us whether someone has evidence of current infection, and along with the right clinical signs and symptoms, can be used to diagnose someone with acute COVID-19 disease.

The primary type of patient that we want to test by a molecular assay is someone who has symptoms that are compatible with COVID-19. If the molecular test is positive in a patient with symptoms, it allows the healthcare team to establish a diagnosis, make important infection prevention and control decisions if the patient is hospitalized, help guide therapy decisions if they are critically ill, or if the patient is an outpatient, the result can reinforce the need for the individual to isolate at home for 10 to 14 days.

But in addition to symptomatic individuals, there’s also been a lot of interest in using molecular tests to identify asymptomatic infections. In other words, using the test to screen those who don’t have symptoms but who may be infected with SARS-CoV-2 and a potential source of infection to others. This type of strategy has led to a significant increase in the demand for molecular testing, which has placed a large amount of stress on the global supply chain for swabs, transport media, test reagents and even equipment.

Even though Molecular PCR have remained the gold standard for diagnosis of COVID-19, due to the ongoing supply chain challenges it’s going to be important to have testing options. So let’s discuss several other testing options including lateral flow immunoassays and lab-based serologic tests. In the months following the beginning of the COVID-19 pandemic, tests that would identify whether someone had developed antibodies in response to SARS-CoV-2 infection gained a significant amount of interest. When a person is infected with a virus, like SARS-CoV-2, their immune system begins to develop a number of proteins, called antibodies that help to fight off the infection and provide future immunity. Typically, the first types of antibodies that are produced are called IgM- and IgA-class antibodies. These antibodies can take between 5-7 days after an infection to develop, and work by binding to the foreign pathogen and flagging it for clearance by the immune system. Later on, usually around 14 days after infection, a different type of antibody, called IgG, is produced. IgG antibodies help to provide longer term immunity against reinfection.

We can use this pattern of antibody responses to help determine whether someone has been exposed to an infectious disease, like SARS-CoV-2. Serology tests work by looking for the presence of those antibodies against a particular pathogen in a patient’s blood. In most cases, a blood sample is taken from the patient, and then is spun down to separate out the part of the blood, which is called serum, that contains antibodies. The serum is then tested and if antibodies are present they are generally detected by a color change on the test.

The early version of COVID-19 antibody tests used a type of technology like a simple pregnancy test. These tests are called lateral flow immunoassays, which work by adding a drop of blood to a test strip, which is usually made up of a substance like filter paper. The blood sample “flows” up the test strip, and if antibodies against the virus are present a colored band appears on the strip. Because lateral flow immunoassays are quick and don’t require special equipment or much training, they’re really well-suited for point-of-care testing outside of the laboratory.

The other class of COVID-19 antibody tests were designed to be more of a lab-based, serologic test. The blood sample is sent to a testing laboratory, where the serum is isolated and prepared for testing. The serum is then added to a test well, and if antibodies against the virus are present, they bind to proteins that are immobilized at the bottom of the test well. A test reagent is added, and if the antibody-protein complex has formed, a color-reaction occurs, which is usually measured by an instrument.

Even though there are similarities between the lateral flow immunoassays and lab-based serology tests, there are some key differences. The lateral flow immunoassays are relatively fast, only taking 15-30 minutes to complete. They typically don’t require any special equipment, so they can be performed in a doctor’s office or in a clinic setting. Some of the early lateral flow assays for COVID-19 were designed to detect IgM antibodies against the virus, so the hope was that they could be used to diagnose patients who were acutely ill. However, we’ve learned that the antibodies against SARS-CoV-2, including those IgM and IgA antibodies, still require 7-10 days to be detectable by most tests, so the IgM-based tests have proved less useful for diagnosing active disease. The lateral flow assays are also less specific, which means they have a greater chance of being falsely-positive. This could mean that a person without COVID-19 might test positive by one of the lateral flow tests.

In contrast, the lab-based serology tests require that the sample be sent to a centralized lab and are generally more accurate. The serum sample is tested on sophisticated equipment, and usually takes multiple hours to complete the test. Most of the lab-based serology tests for COVID-19 have been designed to detect either IgG antibodies, or total antibodies, which consist of a mixture of IgM, IgA and IgG.

There are a few factors to keep in mind for determining the usefulness of a serology test. Unlike the molecular tests, serology assays have limited utility in diagnosing symptomatic patients. This is because by the time most individuals have mounted an antibody response, they are beginning to recover from their illness. However, there are a few situations where serology tests can be useful. First, if an individual has an illness that is highly compatible with COVID-19, but they’ve tested negative by PCR, a serology test could be used to determine whether they have been exposed to SARS-CoV-2. If antibodies against SARS-CoV-2 are detected in these types of cases, it can help the healthcare team determine that the individual’s disease was most likely due to COVID-19. Second, serology tests, especially those that are specific for IgG antibodies, are very useful in helping to identify the number of people in a population who have been exposed to the virus. Testing a large number of people in a community can determine the seroprevalence in that area. In other words, what percentage of a population has been infected with SARS-CoV-2 and generated an antibody response? And finally, antibody tests can be used to identify individuals who have been previously exposed to SARS-CoV-2 and who may be candidates for donating samples that can be used for convalescent plasma therapy.

In summary, antibody tests tell us if an individual has been exposed to the virus at some point in the past, but they don’t tell us when the person was infected.

Recently, there has been a lot of attention on new diagnostic technologies that allow for rapid diagnosis of COVID-19, and those methods include antigen testing. Unlike molecular tests which detect the virus’ RNA that genetic material in clinical samples, antigen tests are designed to detect specific viral proteins. On the surface of the virus, there are a number of proteins, such as the envelope and spike proteins that stick out from the viral envelope. Many of the graphics you see on television for example show the virus as a sphere with these spikes sticking out on the surface, and these are exactly the types of proteins that the antigen tests detect. Antigen assays try to determine whether these proteins are present in a patient’s clinical sample, like a nasal swab.

A few of the main benefits of antigen tests are that they’re relatively easy to perform, and they provide rapid results, oftentimes in as little as 15-20 minutes. A swab of the patient’s nasal passage is collected, and then the swab is typically placed in a small amount of fluid. This fluid is then applied to a test strip, like filter paper, and the fluid slowly wicks up the paper strip. This process is similar to the one I described earlier for lateral flow immunoassays. However, in this case for antigen tests, if viral antigens – or proteins – are present in the sample, they’ll bind to antibodies that have been immobilized on the test strip. A color change occurs, which can be seen as a line on the test strip, much like what you see on a pregnancy test. Again, the results can be available in as little as 20 minutes following collection of the sample, so antigen tests can provide rapid answers.

Although antigen tests have several advantages, there are some limitations to be aware of.  The main limitation of rapid antigen tests is their low sensitivity compared to molecular tests. Historically, antigen tests for other respiratory viruses, such as influenza, have proved to be only 60-80% sensitive compared to PCR-based tests. This means that a significant number of people with the infection may go undetected by antigen tests. Since we don’t have much experience yet with real-world performance of antigen tests for SARS-CoV-2, it remains unclear how well they’ll perform in detecting individuals who are infected with this virus. As more antigen tests become available, we’ll be able to compare their performance to molecular tests and get a better idea of their true sensitivity.

For the most part, antigen tests can be used in similar situations where PCR is appropriate. Antigen tests are typically used to diagnose an individual with an active respiratory infection. This is because the virus has to be present in a patient’s respiratory tract for those specific viral proteins to be detected by the test. However, because antigen tests are typically less sensitive than PCR, the way that we use rapid antigen assays can be different.

Antigen tests may be negative in a patient with lower amounts of the virus present in their respiratory tract, so a negative antigen result doesn’t always ‘rule out’ that the patient has the disease. So the timing of when an antigen test is performed is very important. You want to perform the test when the virus is present at the highest amount in the individual’s respiratory tract, so that you increase the odds of the antigen test being positive. This means that antigen tests for SARS-CoV-2 may need to be performed more frequently compared to PCR. For example, if a patient tests negative by an antigen assay, they may need to be tested again in a few days. Alternatively, some hospitals may have negative antigen results followed up by a PCR test for confirmation.

The strategy of using rapid antigen tests to screen people without symptoms has been proposed and discussed at length by experts in infectious diseases, laboratory medicine and epidemiology. The concept is that if rapid antigen tests were deployed in large numbers to a population, people could be tested frequently, such as every two or three days, to determine whether they’re infected. Using this type of approach, antigen tests would have a higher likelihood of catching someone when they are shedding really high amounts of the virus. The downside, or limitation, of this strategy is that it would require a lot of antigen tests to be manufactured, and the global supply chain challenges that have impacted molecular tests will likely impact antigen tests as well. Also, the more testing that’s performed, the more likely that false-positive test results will occur. This is especially true if testing is performed at high numbers in a population that has a low prevalence of the disease. False-positive results for COVID-19 have real-world implications, including loss of work, separation from friends and family and psychological distress. So these factors need to be carefully considered.

Despite these limitations, rapid antigen tests are a promising new approach to diagnose active SARS-CoV-2 infection, and represent another tool in our diagnostic toolbox.

Reviewing each of these 4 technologies that can be used for diagnosis of COVID-19, you can appreciate that we have the ability to choose among the different options and to rely on complementary information from each of these methods for a better understanding of a particular patient’s situation. Each option should be chosen depending on the clinical information available and by the decision that needs to be made based upon the test result.

Looking forward, we will encounter new scenarios in this dynamic COVID-19 pandemic. As soon as vaccines become available, new types of diagnostics are likely to evolve.

We’re definitely going to continue to see a rapid expansion in innovation of technologies used to screen for SARS-CoV-2 infection, diagnose those with disease, and monitor a patient’s response to treatment. As vaccines become available, it may be important to use existing serologic tests to determine whether certain individuals mount an antibody response and develop specific neutralizing antibodies that can prevent infection. Also, there is a lot of interest in developing tests that can measure a person’s immune response to SARS-CoV-2 infection. After the first week of the disease, we’ve learned that whether a person recovers or progresses to a more severe illness oftentimes depends on how their immune system regulates itself. Those who do poorly often have what’s called a ‘cytokine storm’, where the individual’s immune system gets out of control. Tests to measure a person’s cytokine response may be helpful in managing their disease and determining the best treatment strategies. And finally, as more antiviral medications for SARS-CoV-2 become available, it may be important to not only determine whether the virus is present or not, but how much virus is present before and after starting treatment. Tests that can provide a more quantitative assessment of the amount of virus in a patient’s sample may help us determine whether a treatment is being effective.

It’s going to be an exciting next 6 to 12 months, as we’ll continue to see new types of testing options developed for diagnosis and management of COVID-19.”

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