PCR vs. Antigen Tests

Since emergence of COVID-19 (coronavirus disease 2019, often abbreviated simply to COVID), the virus has been found virtually everywhere in the world and every country is working on ways to test for the disease in the shortest time possible. In Hong Kong, people are used to hearing the government talk about how much people were subject to mandatory testing each day, and this testing in most cases refers to quantitative reverse transcriptase polymerase chain reaction – or RT-qPCR for short. This test looks for part of the virus’ genetic material known as RNA or nucleic acid, so many reports refer to it as a nucleic acid tests. But how does this test actually work? And what are the so-called rapid tests sold in pharmacies, and how do they differ? This article explains the differences between these two common COVID testing methods, how to interpret the test results, and what other testing methods are available.

It All Starts with RNA

Even primary school children known the name DNA, but why has RNA suddenly become a buzzword?

DNA is a double-stranded molecule found in all living things (including animals, plants and viruses), and is essentially a genetic code or blueprint for how these organisms form and develop.

RNA is generally a single-stranded molecule that can replicate, transcribe and transfer part of the genetic code to proteins, so that they can synthesise and perform functions that enable the organism to survive and develop.

The virus that causes COVID (SARS-CoV-2) contains only RNA, so it relies on healthy cells in the body surviving and replicating, and uses its genetic code (RNA) to control and “reprogram” these cells, essentially transforming cells into factories that produce the virus. This is how it infects healthy people and this process is the main goal for every virus.

Identifying and isolating those infected at the earliest possible opportunity is the most effective way to reduce transmission of the virus, so in other words, what we are looking for is the coronavirus’ RNA.

Real Time RT-qPCR

Everyone has seen news reports that refer to the number of confirmed infections. These infections are confirmed by testing samples from suspected infections using the real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) method, so the odds are you have seen this term on a test result.

The main sample collection methods used in Hong Kong are deep throat saliva, and combined nasal and throat swab. The saliva method is the simplest method and is therefore used in many home test kits, but has to be performed in the morning before eating or rinsing the mouth; the combined nasal and throat swab method is user to collect samples when a test report is required, for example in order to travel abroad. Regardless of which collection method is used, the samples are sent to a lab, where similar RT-qPCR test processes are used to test for the virus.

Lab technicians start by removing the proteins and lipids from the sample using a variety of chemical solutions, so that they can extract only the RNA present in the sample. The extracted RNA is a mixture of the body’s own genetic material and any viral RNA that may be present. This process is known as RNA extraction.

Special enzymes are then used to reverse-transcribe the RNA into complementary DNA (cDNA). The technician will then add addition cDNA fragments that complement the reverse-transcribed viral cDNA, and if the sample contains the virus, the fragments will attach themselves to the target regions of the viral cDNA. Some of the added genetic fragments act to build cDNA chains during the amplification process, making it easier to detect the nucleic acid from the virus; and other genetic fragments act to build cDNA strands with fluorescent tags that can then be used to test for short sequences that are typical of the virus.

The mixture is put in an RT-PCR instrument, which cycles between heating and cooling the mixture to cause specific chemical reactions that produce new, identical copies of the target parts of the viral DNA. By running through this cycle many times, the instrument continues to replicate the target parts of the viral DNA, with each cycle doubling the number of copies from the last cycle; two copies become four copies, four become eight, and so on and so forth. A standard RT-PCR process involves 35-40 cycles, with the result that each viral strand present in the sample will produce around 35 billion new copies of the viral DNA fragment by the time the process has finished.

Once the new DNA fragments have been created, the marker tags will attach to the DNA strands and release fluorescent dye that can be detected by the computer in the instrument and displayed in real time on the screen. The computer tracks the fluorescence of the sample after each cycle until it reaches a level that confirms the presence of the virus. The technical will also monitor the number of cycles needed to reach this level, in order to estimate the severity of the infection; the less cycles required to reach the relevant level of fluorescence, the more severe the viral infection is. You have probably seen Dr Chuang Shuk Kwan on TV talking saying that a particular patient had a “Ct level of 17”, for example; this means that the patient’s sample only had to be amplified through 17 cycles in the lab before the virus was detected, and this means that the patient’s viral infection is very severe. By contrast, a Ct value of 37 indicates that it took 37 cycles to amplify the originally miniscule amount of virus to the point that it could be detected, demonstrating that the amount of virus in the patient’s body is relatively small.

While RT-PCR is the best test currently available, it has some drawbacks. PCR tests need to be analysed by experienced, specialist technicians using expensive equipment, and the patient samples also have to be collected by people who have undergone specialist training. The sudden explosion in demand has therefore left medical systems in many countries struggling to cope.

Antigen Tests

Antigen tests are officially known as lateral flow tests (LFT). LFTs are a faster, cheaper way of testing for COVID-19. Once the swab is removed from the back of the nose or throat, it is mixed with an extraction solution, after which a few drops of the mixture are placed into a well in a small device, and the liquid is then absorbed on a reagent paper test strip and enters the device. If COVID-19 proteins are detected, the second line [on the screen/on the test strip] will show not only that a test is in progress, but also a red line. It works a little like a pregnancy test, but looks for antibodies that bind with COVID-19 proteins, rather than a particular hormone.

When is it appropriate to use LFTs?

Some countries have recently announced that they will provide all adults with two LFTs per week with the goal of screening out any COVID cases that might have been missed as soon as possible. Finding such cases, for example those with no symptoms, will help to prevent transmission of the virus. While the Hong Kong government has established a number of free test centres that provide PCR testing, the long incubation period of the COVID-19 virus means that it is not possible to perform daily tests. If residents such as students and others who need to meet with others regularly could also perform two LFTs per week themselves, it would effectively prevent transmission of the virus. The advantages of LFTs are that they are cheap, the results are available within 30 minutes, and they can be performed at home.

How accurate are LFTs?

There are two issues that can occur with regard to accuracy:

  • False positive: the test falsely indicates that a person has COVID
  • False negative: the test fails to detect that a person is infected with the virus

Data from the UK’s Department of Health and Social Care comparing LFTs with PCR tests, indicates that LFTs have a false positive rate of less than 0.1%, but a false negative rate as high as 50%, while the ability to take the sample correctly was critical to the results.

How to choose the right test method for you

If circumstances permit, RT-PCR remains very much the gold standard for testing, as it can detect the genetic material of the virus (RNA, nucleic acid) even in the early stages of infection. However, the relatively high cost means that combining it with LFTs is the best option when frequent testing is required.

Antigen tests (LFTs) are simple and quick

Antigen tests are the simplest and quickest of the two testing methods explained above. The goal of testing is to find recognisable proteins or nucleic acids from foreign bodies such as bacteria or viruses, i.e. antigens. Antigen testing is also used for other diseases such as flu and does not require expensive instruments or specially trained test technicians; the results are also available in 15-20 minutes.

Our new CovidCare Rapid COVID-19/Influenza A+B Combined Test Card can be used to conduct rapid qualitative assessment of SARS-CoV-2 virus antigens and influenza A/B antigens in the mouth, nose or throat. Oral or nasopharyngeal swab samples from the test subject can be rapidly and effectively tested for the presence of antigens for COVID-19 and the most common influenza A and B strains in Hong Kong. During the early stages, COVID-19 and influenza can present with symptoms that are similar to the common cold, or even with no symptoms at all, so patients can only tell if they are infected by being tested for the viruses. This single test looks for three different viruses, allowing patients to discover their condition and seek medical attention at the earliest possible opportunity; you can get treated as soon as symptoms occur, helping you to avoid serious symptoms or even death.

You can get tested in the safety of your home by following the simple six-step process explained in the instructions. The results will be available in 15-20 minutes, so you don’t need to queue up at a test centre for a specialist to collect a sample, or wait for the lab test results to arrive; this also means that you can avoid the risk of infection when passing through crowded areas.

If your test result is positive, please consult your doctor immediately.