Many diagnostic tests for COVID-19 are available so far. In this blog entry we are going to show the main characteristics and the main advantages and limitations for detecting, screening and monitoring the disease.
- 1) Background
- 2) Reverse-Transcription Polymerase Chain Reaction (RT-PCR)
- 3) Lateral Flow / Colloidal Gold Immunochromatography
- 4) Enzyme-Linked Immunosorbent Assay (ELISA)
- 5) Conclusions
A range of molecular techniques ranging from laboratory testing to point-of-care tests are under development or already available for the diagnosis and management of COVID-19 patients. These techniques, while well-known to researchers and clinicians, as well as antibody and antigen assays, may be relatively unknown to the broader community. This blog entry was written to outline and explain the basic principles of the main COVID-19 diagnostic tests currently in use.
2) Reverse-Transcription Polymerase Chain Reaction (RT-PCR)
PCR is a very common scientific technique that has been widely used in research and medicine for around 20-30 years to detect genetic information. RT-PCR is a special version used when RNA is being detected and it is now being used as a test to detect SARS-CoV-2. This type of test has frequently been used as a frontline test for COVID-19 as it directly tests for the presence of the virus RNA.
RT-PCR tests are fairly quick, sensitive and reliable, capable of producing results in 3-4 hours, although this usually takes longer if samples must first be sent to specialised external laboratories (6-8 hours on average). Many diagnostic and research companies produce RT-PCR products, tests and machines so the technology is widely available. Some RT-PCR tests are developed as an `all in one’ kit, reducing laboratory handling and potential for contamination.
Once a sample has been collected, chemicals are used to remove any proteins, fats and other molecules, leaving only RNA behind. This will be a mixture of a person’s genetic material as well as any viral RNA that might be present.
The test kit enzymes copy the RNA to DNA, which is amplified to allow virus detection by using a PCR machine which cycles the test temperature so that roughly 35 billion copies of viral DNA are made for each viral RNA strand that was originally present.
Fluorescent markers are typically used to bind to the amplified DNA and produce light, which can be read by the machine to produce the test result. If the intensity of the light produced within the sample reaches a certain threshold, this is classed as a positive test. The number of PCR temperature cycles that were required before the fluorescence threshold was reached is recorded and gives an estimate of how much virus was present in the patient sample.
In the first step, viral DNA is heated, separating its two strands. The reaction is cooled to 55ºC which causes the separated two strands to bind with small complementary stands of DNA in there as well as fluorescent markers. Heating the reaction up to 72ºC allows the new stands of DNA to bind and extend producing 2x the amount of DNA compared to that in step 1. The heating and cooling cycles (steps 1-3) are repeated around 30 times, each time doubling the amount of viral DNA that forms. This can be detected by the instrument.
What does the test detect?
RT-PCR detects whether or not viral RNA is present in samples from a patient. It does this by capturing and amplifying regions of the virus’ genetic material, usually the Spike protein, N protein or Envelope. To measure the viral RNA, it is converted to DNA, copied many times using repeated temperature cycles in a PCR machine and then fluorescent markers are used to detect the virus. If the amount of fluorescence goes above a certain level, this confirms that the virus is present. The number of temperature cycles the machine performs to reach this threshold is recorded to estimate how much virus was present in the patient sample. The lower the number of cycles, the more virus was present.Commonly these samples are taken from the nose or throat using either long or short swabs, but samples can be collected in other ways too. Collecting samples from where the virus is shedding or multiplying, improves the accuracy of the test.
What does the result mean?
An RT-PCR test is highly sensitive and fairly reliable if performed on a sample from an infected part of the body whilst an active infection is occurring.
Positive test result: – A positive PCR result means that the person the sample was taken from is currently infected by the virus.
Negative test result: – A negative PCR result could mean that the person is not currently infected by this virus, the virus is not present at the site the sample was taken from, the sample taken was of poor quality, or that it is too early, or too late in the infection to detect replicating virus. This is why negative test results require new patient samples to be taken a few days later to reduce the chance of incorrectly missing an infected person.
The RT-PCR test cannot detect if a person has had the virus and then cleared it after the end of the COVID-19 disease, i.e. whether a person had the disease, as it only detects when active virus is present.
Advantages and Limitations
- – RT-PCR is a robust and well documented technique.
- – With RT-PCR being so common in research and medicine, the technology is already in place to test for COVID-19.
- – RT-PCR can detect current infections of disease, allowing medical staff to determine who is currently infected and who is not.
- – RT-PCR relies on capturing and detecting the virus and so it is possible to miss patients who have cleared virus and recovered from disease.
- – The distribution of virus across the respiratory tract varies between patients, so even if a person is infected, the virus may only be detectable in sputum or nasopharyngeal swab but not necessarily at both locations at the same time.
- – RT-PCR for COVID-19 can only tell if a person is currently infected with this particular coronavirus. It can’t provide information on other diseases or symptoms.
3) Lateral Flow / Colloidal Gold Immunochromatography
Lateral flow assays have commonly been referred to as ‘Rapid tests’ in the media as they are currently used to detect antibodies to disease in a patient’s blood. The technology is also being tested for antigen use too. Lateral flow assays use the same technology commonly used for pregnancy tests. Lateral flow tests can detect antibody to virus from patient blood indicating that the patient has COVID-19 or has recovered from COVID-19. Less commonly, lateral flow tests can be used to detect the presence of active virus by detecting virus proteins directly.
Antibody lateral flow tests for SARS-CoV-2 are produced as test kits used by a specialist or clinician rather than by patients themselves. They require a drop of patient blood, either from a vein or from a small finger prick, similar to a finger prick test used for blood sugar monitoring in certain types of diabetes. These types of tests work very differently to RT-PCR techniques and detect the patient’s immune antibody response to the virus rather than detecting the virus itself.
How it works?
Lateral flow immunoassays for COVID-19 are simple devices that can detect antibodies in the blood.
A small sample of patient blood is taken from a vein or from a finger-prick by a clinician and dropped onto a spongey pad within the test device. A few drops of a diluting liquid called a ‘buffer’ are added to help the blood sample flow across the device. As the sample moves through the device, antibodies against SARS-CoV-2 that are present in the sample will attach to chemicals in the device, capturing the antibodies on the test and control lines. This capturing and binding process results in a colour change along the test and control lines which can be seen by eye, producing one, two or three lines depending on the type of antibodies are present (IgM or IgG).
What does the test detect?
Lateral flow immunoassays for SARS-CoV-2 detect two types of protective antibodies that are produced by the body when the immune system recognises a foreign structure, in this case SARS-CoV-2, the virus causing COVID-19. These antibodies help fight the disease and remain in the blood for months after the virus and disease is cleared. The presence of antibodies in the body is often referred to as immunity or that a person is immune to a virus, as these antibodies protect against re-infection and return of the same disease.
When we are infected by virus, our immune system produces early `prototype’ antibodies (IgM) with intermediate strength binding to virus, that are able to start working to clear virus about 5 days after a new infection. Typically at 8 to 10 days after infection, IgG antibodies with high binding strength, can work to help more rapid virus clearance. Antibodies act by developing a matching contoured surface to stick to foreign antigens, using a sophisticated selection process to amplify antibodies with the best surface match and strongest binding.
What does the result mean?
Antibody lateral flow immunoassays detect antibodies to the virus in the blood. They don’t detect the virus itself. The ability to detect the immune system response reliably using only one sample (blood) is a huge advantage, as is the amplification of the detection signal generated by the body immune response. Using the antibody response alone does not allow distinction between individuals who are currently infected and those who have cleared the virus infection. Antibody tests provide a hugely important ability to detect past infection with virus to identify people who were asymptomatic, people who have cleared the virus and so no longer risk being infected or spreading the virus to others. In addition, antibody tests are critical for assessing population spread of the virus and the level of ‘herd’ immunity in the population. This is important for understanding the potential consequences of lifting or enforcing measures to control the virus such as quarantine, social distancing, school and workplace closures.
The antibody IgG and IgM lateral flow immunoassay tests are very simple to read: A control line must appear to show that the assay has worked correctly. Then, test lines will appear if either of the antibody types are found in the sample. The appearance of lines for IgG or IgM, or both indicate a positive test – showing that the patient has been infected with the COVID-19 coronavirus.
Advantages and Limitations
- – Lateral flow assays are extremely quick per patient, giving results in just 15 minutes.
- – Testing levels of antibody in blood allows a single patient sample from one accessible part of the body where sampling is non-invasive to be tested for presence of virus.
- – Lateral flow tests are more expensive and time consuming for large batch testing than specialist laboratory based antibody tests such as ELISA.
- – So far, available lateral flow tests can only determine if a patient has at some point been infected with COVID-19. Further testing would be needed to check if a patient is currently infected. Future versions of this technology might allow clinicians to detect current infections.
4) Enzyme-Linked Immunosorbent Assay (ELISA)
An Enzyme-Linked Immunosorbent Assay (ELISA) is a common biochemical technique that can be used to detect antigens or antibodies, depending on the type of test used. ELISAs use enzymes linked to antibodies that can attach to the molecule that is being tested for and cause a colour change that can be measured by a specialised machine. The strength of the colour change gives scientists and clinicians an idea of the number of molecules of interest in the sample. ELISAs can be done as standard batches of up to 96 assays completed at the same time, allowing cheap and time effective method for batch testing of large numbers of patient samples at the same time. This technology could help speed up the number of patients that can be tested for SARS-CoV-2. The most effective ELISA assays in monitoring prior infection detect antibodies against SARS-CoV-2. Future ELISA could be used to test for active virus infection by detection of virus protein (antigen) testing, but this testing will not be as accurate and is as yet unproven.
How it works?
An ELISA detects antibodies produced in patient blood due to infection with SARS-CoV-2. The whole test can be performed in one tube or well and involves mixing patient samples, antibodies, antigens and enzymes together with a colour changing molecule. The example below describes a typical antibody ELISA test.
- A patient sample of plasma is added to a well containing SARS-CoV-2 specific antigen.
- The patient antibodies to SARS-CoV-2 stick to the SARS-CoV-2 proteins coated on the bottom of the welland the rest of the liquid sampleis washed off.
- Other laboratory-produced antibodies with enzymes attached are added and stick to the patient antibodies present in the well as a “second layer”. Excess antibody with enzyme attached is washed off, so enzyme is only present in wells when a patient has produced antibodies to COVID-19.
- special colourless molecule is added to the well.
- In wells containing samples from patients who have been infected with COVID-19 and so have antibodies to the virus, the “second layer” antibodies with enzyme act on the special colourless molecule causing it to rapidly change colour indicating a positive result.
- The colour change can be read either by eye or by a machine called spectrophotometer.
- If the patient had not been infected with COVID-19, the enzyme-linked antibody would not stick to anything in the well and te colourless molecule would not change colour.
What does the test detect?
ELISA tests to detect antibodies are detecting the antibody response to COVID-19 infection. Detecting antibodies to SARS-CoV-2 could tell a clinician if a patient has been infected with COVID-19, either currently or in the past. However infected patients will not be detected immediately on infection, but only when the immune system to the virus can be detected in blood, roughly 5 days after infection for a test detecting IgM antibodies, which is about the same time that symptoms occur. Current knowledge suggests once a person has been infected with virus, their immune system will prevent a future infection with the same virus. This antibody ELISA test provides very important information for diagnosis, management and recovery from COVID-19 and will also help researchers evaluate how many people in the population have been infected, which is important to planning infection control.
What does the result mean?
An antibody test using ELISA would show a positive result (colour change) if the patient has antibodies to COVID-19. This might not mean that they currently have the virus, only that they have had it at some point. This is because antibodies stay in the blood even after the infection is gone to help provide the body with immunity if they come into contact with the virus again. A negative result (no colour change) would mean that the patient has not been infected with COVID-19 and may have no immunity against it.
ELISA antigen tests may be developed in the future to detect current infections. Such an antigen test using ELISA would show a positive result (colour change) if a patient has COVID-19 in their blood. This would indicate that the patient is currently infected with the COVID-19. A negative result (no colour change) would indicate that no COVID-19 antigens were found in the patient’s sample. This could mean that the patient does not have COVID-19, but might also mean that they are too early in their infection to be positive. If they have symptoms of COVID-19 they should be tested again a few days later to make sure.
Advantages and Limitations
- – ELISA is a simple and cheap laboratory technique.
- – ELISA is well established and documented within science and medicine.
- – Results can typically be produced within 1 to 3 hours of collecting a patient sample.
- – Because it is so quick to perform it can be done in a hospital laboratory, cutting down the time to diagnosis. ELISA testing can be completed on multiple samples at once, so it can be used for rapid testing scaled up to test larger numbers of patients.
- – The starting material, plasma or serum, should be obtained from invasive methods (venous blood)
- – Four main types of tests are being used or being developed to test for SARS-CoV-2, the virus causing COVID-19.
- – These tests are at different stages of development, validation and production.
- – Each test type has its own distinct advantages and disadvantages inherent to the underlying technology.
- – A combination of testing types used at different times may be useful for patient management and population pandemic control of COVID-19.
|Technology||Molecule tested||Laboratory or Point-of-care||Time to results||Sample||Number of samples per batch|
|RT-PCR||viral RNA||Laboratory||3-4 h||nasopharyngeal swab, sputum||Up to 96 samples|
|Lateral flow||Antibody||Point-of-care||15-20 min||Blood||1 sample per assay|
|ELISA||Antibody||Laboratory||1-3 h||Blood||Up to 96 assays|