Every day there’s a new headline about the best ways to test for SARS-CoV-2 virus, the virus that causes COVID-19. In the following Q&A, Joshua Trotta, Senior Director Business Development, discusses what you need to know about qPCR, the gold standard for COVID-19 testing. This is a follow-up to our “How does a COVID-19 test work?” interview with Joshua on Science with a Twist earlier this year.
What is an RT-qPCR test?
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“PCR” is shorthand for “polymerase chain reaction,” a scientific methodology to make millions and billions of copies of a specific DNA sample, which for the past 25 years has been one of the most widely used techniques in molecular biology.
The genetic material in SARS-CoV-2 virus is stored in RNA. In the case of COVID-19 testing, RNA from a SARS-CoV-2 sample is first converted to its complementary DNA sequence by a process called reverse transcription (RT). The DNA thus transcribed is amplified by qPCR, where “q” stands for “quantitative”; hence the technique is called RT-qPCR[1].
RT-qPCR can detect the genetic information (RNA) of the novel coronavirus—even if the virus is present in extremely small amounts. Researchers estimate that RT-qPCR can detect as little as ~1,000 copies of viral RNA per milliliter, or 10 copies per analytical limit of detection (LoD). This means it is possible to diagnose someone as positive for COVID-19 even when their sample contains a very small amount of virus.
This means that COVID-19 RT-qPCR tests are not only able to detect the virus’s genetic information, they are also able to quantify the amount of that genetic information that is present in a sample. The Applied Biosystems TaqPath COVID-19 Combo Kit from Thermo Fisher Scientific is designed to detect the presence of the virus at very low levels and uses an algorithm to provide a positive or negative result.
How is a SARS-CoV-2 sample collected for RT-qPCR testing?
Samples can be collected in a few different ways. To test if someone with COVID-19 symptoms is infected with SARS-CoV-2, a health official might collect cells from the person’s nose and/or throat usually by using a nasal, throat or nasopharyngeal swab. Alternatively, a saliva sample can be collected.
As organizations look for routine testing solutions in workplaces and universities, they are also exploring options for self-collection. These self-collected samples are then sent to a lab where the RT-qPCR test is run, which takes about two to four hours to complete.
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What is an antigen COVID-19 test?
While an RT-qPCR COVID-19 test detects the genetic material of the novel coronavirus, an antigen-based COVID-19 test is designed to detect something different: proteins on or within the virus.
For someone getting an antigen COVID-19 test, the sample is collected in the same way as for the RT-qPCR COVID-19 test—usually by nasopharyngeal swab. However, in the case of an antigen COVID-19 test, the sample does not need to be sent to a lab for analysis. Instead, it can be analyzed at the point of care in a process that can take as little as 15 minutes.
The trade-off for the speed and convenience of an antigen COVID-19 test is specificity. The antigen COVID-19 test requires a higher viral load in the sample to yield a positive result. As a result, someone who tests negative with an antigen test may actually have an active COVID-19 infection, but there are too few virus proteins present in the sample to register positive on the test. That same person my take an antigen test the next day and get a positive result, simply because they have more of the virus in their system.
What are the main differences between the different types of COVID-19 tests?
When it comes to COVID-19 testing, your choice of test type usually is a choice between accuracy and speed. You need to fully understand the tools available in the toolkit, and then pull out the right one at the right time.
A COVID-19 antigen test can provide results quickly, but RT-qPCR is considered the gold standard for COVID-19 testing as it provides a more definitive answer as to whether an individual has the virus in their body.
In fact, in situations where antigen testing is used for rapid screening, negative results are often sent to the lab for confirmatory RT-qPCR testing to verify if the individual is indeed virus-free.
In addition, there is a third type of test: a COVID-19 antibody test. An antibody test is a blood test that does not detect active virus. Instead, it detects antibodies to SARS-CoV-2. The presence of antibodies indicates that someone is actively dealing with, or recovered from, a COVID-19 infection.
What factors can lead to a false-positive or false-negative test result?
It’s also important to understand that, like any other diagnostic test, the results of COVID-19 tests can be impacted by sample collection. The reliability and accuracy of these tests depend on:
- How the sample was collected—it is always recommended to follow the manufacturer’s Instructions for Use (IFU) on the proper use of the test and approved sample collection methods.
- How many samples were collected—for nasopharyngeal and nasal swabs, samples from both nostrils are recommended.
- When the sample was collected—for example, someone who is newly infected may not have detectable amounts of virus in their system.
- How long ago the sample was taken—samples should arrive at the lab within 72 hours after collection.
- How the sample was stored before analysis—again, it is important to follow the manufacturer’s IFU, as extreme temperatures or other harsh conditions can damage viral molecular integrity.
What lies ahead for COVID-19 testing?
Health officials predict an increase in SARS-CoV-2 infections and they continue to weigh the pros and cons of different testing methodologies and protocols.
qPCR testing is going to become even more important given its ability to detect SARS-CoV-2 even when it’s present in extremely small quantities, like when someone has been recently infected and has a relatively small viral load.
With qPCR, it’s possible to make a diagnosis early on in the infection, meaning the infected individual can go into isolation earlier to prevent passing the virus on to others. Until a vaccine is widely available, testing is the key to stopping this crisis.
Additional resources
- COVID-19 testing products
- Real-time PCR Handbook
TaqPath COVID-19 Combo Kit: For Emergency Use Authorization (EUA) only. For prescription use only. For in vitro diagnostic use.
[1] Bustin SA, Nolan T. RT-qPCR Testing of SARS-CoV-2: A Primer. Int J Mol Sci. 2020;21(8):3004. Published 2020 Apr 24. doi:10.3390/ijms21083004
