Everything you need to know about RT-PCR Covid-19 testing

Even though Covid-19 testing has become a familiar part of life for many of us over the past year, there’s still a lot which is often misunderstood about the different types of test on offer and how they work.

Not all Covid-19 tests are equal. Even the commonly recognised PCR test, which is used by the NHS, has a range of testing method variations used in laboratories.

So, what are the differences, why do scientists favour PCR testing and how do the tests actually work?

We’ve gathered some of the key questions, and answers, to help explain the process of Covid-19 testing in more detail.

What does PCR stand for?

PCR stands for polymerase chain reaction. It is the shortened name for a particular method of scientific testing used to diagnose the presence of a virus. In this case, Covid-19, but the method can be, and is, used routinely to test for other types of infectious disease such as Ebola and Zika.

What about RT-PCR?

RT-PCR stands for reverse transcription polymerase chain reaction.

You don’t always see the letters RT before PCR when reading about Covid-19 tests. This is because reverse transcription is an additional process that isn’t included in all PCR tests. And, often, RT is left off simply in the interest of simplifying the name when talking about Covid-19 testing.

This meant in the early days of COVID testing, Laboratories were issuing results which were actually RT-PCR tests, but the result said simply PCR test. These could sometimes get rejected by airlines, despite actually being truel RT-PCR tests for Covid. Thankfully, all laboratories (that we use at least!) now include the full RT-PCR term for the COVID test.

Not all viruses and pathogens are the same. Some already contain the deoxyribonucleic acid (DNA) genetic information needed for testing to work. Others, like Covid-19, contain only ribonucleic acid (RNA).

RNA needs to be translated into DNA before the test can identify the presence of the virus. The additional reverse transcription (RT) step is needed to translate the RNA into DNA.

Is PCR testing the only way to test for Covid-19?

PCR testing alone isn’t effective for diagnosing Covid-19. Because the Covid-19 virus contains only RNA, the additional RT-PCR process is needed in order to trigger the reaction that detects the virus.

There are also rapid result options which use a different testing methodology. These include lateral flow testing. Although lateral flow tests deliver faster results than PCR tests, there are concerns about the accuracy of this method.

You may have also heard about antibody testing. Unlike the other methods which are designed to diagnose if you have Covid-19, antibody tests are designed to tell you if you have had Covid-19 previously.

How does the RT-PCR test actually work?

RT-PCR testing involves collecting a sample from the nose and throat using a swab. This sample is then sent to a laboratory for testing, where the RNA is isolated and applied to other ingredients that recognise and bind to SARS-CoV-2. The following reaction, or lack thereof, is used to diagnose the presence of the virus.

Once the sample is received at the laboratory, it is treated with chemical solutions to strip away proteins and fats, extracting only the RNA that is present. This RNA contains a mix of the person’s own genetic material and, if present, the virus’s RNA.

Using a particular enzyme, the RNA is then reverse transcribed to DNA using a particular enzyme. After this, scientists add short fragments of complementary DNA to specific sections of the transcribed viral DNA.

If the virus is present, the fragments attach to target sections of the viral DNA. Some of the added genetic fragments are used for building the DNA and adding marker labels to the strands. These marker labels are used to detect the virus.

The mixture is placed in an RT–PCR machine, intermittently heating and cooling in order to trigger chemical reactions that create identical copies. A standard RT–PCR sequence usually goes through 35 cycles. At the end of the process, around 35 billion new copies are created from each strand of the virus present in the sample.

As copies of the viral DNA sections are made, marker labels attach to the DNA strands, releasing a fluorescent dye. The machine’s computer measures the reaction, tracking the amount of fluorescence after each cycle. Presence of the virus is confirmed when a certain level is reached.

Scientists also measure the severity of the infection by looking at how many cycles it takes to reach this level. The fewer the cycles, the more severe the infection is.

How accurate is this testing method?

The RT-PCR testing method uses proven primer and probe sequences targeting two different genes from SARS-CoV-2 (assay N1 and assay E). This represents the next generation of testing capability and reduces the risk of false positive result.

It also includes an internal human control (assay RP). This internal human control ensures that a sufficient quantity of human genetic material has been collected during the sampling process and reduces the risk of false negatives from poorly sampled or unused swabs.

The primer and probe sequences have been previously defined by the US Centre for Disease Control (CDC) and the World Health Organisation (WHO) and have been extensively validated for the detection of Covid-19. It is a highly accurate process, with a test specificity of 100% and a test sensitivity of over 95% at 0.75 copies of vital RNA/ul

Why is the TR-PCR method used?

The RT–PCR technique is both highly sensitive and specific. Compared to other available virus isolation methods, real time RT–PCR is significantly faster and has a lower potential for contamination or errors. It continues to be the most accurate method available for the detection of the Covid-19 virus.

What do the results of the test look like?

After your sample is processed, the results are quickly emailed to you as a laboratory certificate, containing a positive or negative result, and information about the test. Here is an example of the results.

About the Blue Horizon laboratories

The standard of test results is critical, and our partner laboratories function to the highest clinical and administrative criteria. Laboratory tests and results are subject to stringent quality control processes and are assessed to ensure the highest levels of precision and performance.

Our partner laboratories includes the largest independent supplier of medical laboratory diagnostic services from the United Kingdom. It has a proven reputation for safe and effective specimen handling and provides blood testing solutions to clients including clinicians, businesses, private hospitals.

Find out more about the RT-PCR test and order online.