Testing for COVID-19: How it Works

Depending on your viewpoint, source of information and tolerance for risk, this can be a frightening time for persons all over the planet. The level of disruption to daily life that we’re all experiencing due to COVID-19 is unprecedented.

We are all either not working, working from home and away from our normal offices, or in some cases working many more hours to cover for sick coworkers and caring for SARS-CoV-2-infected persons.

But there is good news if you find that information is power. We hope that some information about the testing being used in the US for this novel coronavirus might be fuel for you, empowering in terms of information.

What is the Name of the Virus, and the Disease?
Since this is a global pandemic, the World Health Organization was instrumental in naming the virus and disease. From this web page: the disease is called COVID-19.

The coronavirus responsible for this disease is SARS-CoV-2.

What Test is Used for COVID-19?
The test that Centers for Disease Control (CDC) is using for COVID-19 as of March 17, 2020, is: “Centers for Disease Control and Prevention (CDC) 2019-Novel Coronavirus (2019-nCoV) Real-Time Reverse Transcriptase (rRT)-PCR Diagnostic Panel”. In other locations and in some labs in the US commercially-produced tests or laboratory-developed tests (LDTs) may also be used.

What is RT-PCR?
rRT-PCR* is a real-time, Reverse Transcriptase Polymerase Chain Reaction in which amplification is actively monitored as the reaction progresses. This is a fast, convenient and quite sensitive test, meaning that it can be run in a short 1–2 hours, on samples that contain potentially very small amounts of RNA. However, because of the speed and sensitivity, there is potential for error if the test is not run in a highly clean and controlled environment with carefully produced reagents. So, rRT-PCR must be run in a lab that meets certain guidelines, including maintaining an environment that is free from extraneous nucleic acid contamination.

Testing for COVID-19 starts with collection of a nasopharyngeal swab (by medical professionals) from which RNA is isolated and tested in RT-PCR for the presence of viral nucleic acid.

What samples types are collected for COVID-19 testing?
As of March 13, 2020, upper respiratory nasopharyngeal swabs are recommended for testing, as specified by CDC. CDC specifies not only the swab type, but also storage and transport time and temperature requirements. Nasopharyngeal swab samples are taken and collected by medical professionals in a clinic, or in some cases, at a drive-up collection site.

We have a swab. What’s next?
The swab is placed into a special transport medium and sent to a testing laboratory—at this time these laboratories are often the State Health Labs here in the US—these labs have the test kits provided by CDC for use in COVID-19 testing. The swab is tested for the presence of SARS-CoV-2 RNA (ribonucleic acid).

What is RNA?
RNA is a nucleic acid (as is DNA, deoxyribonucleic acid) found in all or almost all living things. One exception to this is viruses. Some viruses contain DNA encased in a protein structure, while other viruses, like SARS-CoV-2, contain RNA encased in a protein structure. RNA is in all of our cells, and while it’s composed of the same four nucleotides no matter where the RNA came from, the arrangement of those nucleotides is unique to each organism, virus to human.

Since RNA is constantly shed through things like hair and skin cells, it is a potential contaminant to laboratory tests. COVID-19 tests must avoid any and all extraneous RNA. Thus the laboratory that does this testing must meet certain regulatory requirements, and be operated under the strictest standards, including being free of extraneous nucleic acids, both DNA and RNA. The reagents used in the test kits must also be free from contaminating nucleic acids. In addition, testing must be performed by specially-trained personnel. Testing is only done by laboratories that can maintain these strict standards of training and cleanliness.

More on rRT-PCR
Very small, nanogram amounts of RNA can be isolated from a nasopharyngeal swab and used in rRT-PCR (this is why we say that the rRT-PCR test is sensitive). During rRT-PCR, the RNA is copied by the reverse transciptase (reverse transcribed) to produce a matching piece of DNA called a cDNA (copy DNA). This cDNA, in turn, serves as a template for PCR to make enough copies to analyze (amplification). In addition to reverse transcriptase, other reagents used in rRT-PCR are primers, nucleotides (the building blocks of DNA) and a fluorescent dye.

The amplified DNA can be compared to positive control and negative control samples included in rRT-PCR and on the same plate. These controls ensure that: 1) the rRT-PCR run performed as expected and was free of contaminants, and 2) the amplified DNA is truly from SARS-CoV-2.

rRT-PCR is run on an instrument that can process plates containing multiple RNA samples, plus controls. The fluorescent dye in each plate well allows the instrument to monitor, during the rRT-PCR, the presence of and amount of DNA in each well.

How is the test run?
In the US, CDC recommends that the rRT-PCR test, “is intended for use with the Applied Biosystems 7500 Fast DX Real-Time PCR Instrument with SDS 1.4 software.” However, outside of the US, other instruments are being used for COVID-19 testing. Instruments like this can run multiple tests and samples at one time, safely and quickly.

Thoughts on the Challenge Ahead
As SARS-CoV-2 testing continues to ramp up in the coming days, we can look forward to an expansion of testing and thus test results, an important step forward in meeting the challenge of COVID-19 disease. We may see a rise in positive cases, but this is progress, and an essential step in halting the spread of this disease. Knowledge is power.

Learning more about this disease and it’s spread, via testing, is a challenge, no doubt. Together we can meet this challenge and move beyond it.

*Due to differences within scientific literature, there are several acronyms describing PCR technologies utilizing reverse-transcriptase and offering real-time quantitation of DNA. The acronym implemented by the CDC, rRT-PCR, is equivalent to Promega RT—qPCR (Reverse Transcriptase quantitative Polymerase Chain Reaction).

Additional Information
This recent blog has more information on how Promega is supporting scientists doing COVID-19 research.

CDC SARS-CoV-2

WHO Corona Virus Site

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Kari Kenefick

Kari has been a science writer/editor for Promega since 1996. Prior to that she enjoyed working in veterinary microbiology/immunology, and has an M.S. in Bacteriology, U of WI-Madison. Favorite topics include infectious disease, inflammation, aging, exercise, nutrition and personality traits. When not writing, she enjoys training her dogs in agility and obedience. About the practice of writing, as we say for cell-based assays, "add-mix-measure".

7 comments

      1. Hi there,
        Our technical services team would need a few more details to give you an answer. If you are referring to the CDC protocol, in the US CDC recommends that the rRT-PCR test, is intended for use with the Applied Biosystems 7500 Fast DX Real-Time PCR Instrument with SDS 1.4 software. However, outside of the US, other instruments are being used for COVID-19 testing. We do have a CDC protocol here: https://www.fda.gov/media/134922/download. If you have more questions or want to speak with a scientist we would be happy to help: https://www.promega.com/support

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