RT-qPCR diagnostic tests for SARS-CoV-2 – an overview

by | 20. 11. 2020 | Inside laboratory

Reading Time: 7 minutes

The COVID-19 pandemic is turning our lives upside-down and we at BioSistemika want to contribute what is in our power to help overcome it. In this article, we have summarized some basic information regarding SARS-CoV-2 diagnostics, including quantitative RT-PCR (RT-qPCR) protocols, troubleshooting RT-qPCR, and ideas for improvements. This information should give you a brief overview of the current situation in COVID-19 diagnostics and provide tools for improved management of increasing testing rates.

COVID-19 is caused by a virus called SARS-CoV-2 and can cause a respiratory illness that displays in a wide array of symptoms, from mild upper respiratory symptoms to severe progressive respiratory failure. The disease can, in the worst cases, lead to death (WHO, 2020). Its genome was first sequenced in January 2020 and is approximately 30 kb long. More details about the SARS-CoV-2 genome structure can be seen in Figure 1.

Schematic representation of SARS

Figure 1: Schematic representation of SARS-CoV-2 genome. SARS-CoV-2 contains a positive-sense single-stranded mRNA genome with a 5′ capped mRNA sequence (C) and a 3′ poly-A tail. The coding genes are: ORF1a, ORF1b, Spike (S), ORF3a, ORF3b, Envelope (E), Membrane (M), ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF14, Nucleocapsid (N), and ORF10 (D’Cruz et al., 2020).

As SARS-CoV-2 is highly infectious it is important to perform fast and reliable tests to understand how the pandemic is spreading. We can assume how many people are infected by the number of positive tests, yet we cannot say that the number of positive tests is the number of all people who have COVID-19. Therefore, it is important to perform an adequate number of tests that give reliable results. As of November 16th, there were more than 320 million COVID-19 tests done in the USA and China, approximately the same number in the EU, followed by more than 100 million in India (Our World in Data, 2020).

World Health Organization (WHO), as well as Centre for Disease Control and Prevention (CDC) and European Centre for Disease Prevention and Control (ECDC), agreed on the main types of SARS-CoV-2 detection tests:

  • Nucleic acid amplification tests (NAAT) are tests that are based on RT-qPCR and detect the presence of SARS-CoV-2 viral RNA.
  • Antigen tests are tests that detect the presence of viral antigen that is typically a part of a surface protein. Existing COVID-19 rapid tests are based on antigen detection.
  • Antibody tests are tests that detect the presence of antibodies that are produced against the SARS-CoV-2 virus. The most widely used serologic assays for SARS-CoV-2 are ELISA (enzyme-linked immunosorbent assay), CLIA (chemiluminescence assay) and LFA (lateral flow assay).
  • Other upcoming test options: Isothermal Nucleic Acid Amplification, RT-LAMP, RPA, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), Cas13a Assay, Next Generation Sequencing, etc. (D’Cruz et al., 2020)

RT-qPCR detection of SARS-CoV-2

When the COVID-19 pandemic started, there was an immediate need to consistently perform large numbers of tests. In the current situation, we are mostly relying on molecular diagnostics with RT-qPCR, which provides us with quantitative results of SARS-CoV-2 presence in the specimen.

Usage of RT-qPCR can produce fast and reliable results in big quantities and that is one of the main reasons why it is so widely used in the COVID-19 pandemic. In 3-4 hours, one batch of 96 or 384 samples (triplicates for one patient) with controls can be done. Testing sensitivity of an RT-qPCR test for COVID-19 is estimated at 95% and RT-qPCR can detect the presence of the virus at very low viral titers (<10 copies/reaction). That is of crucial importance as fast detection of infected individuals is the main priority to obtain public health and preventing viruses to spread out of control (Younes et al., 2020).

With RT-qPCR tests, we detect the genetic material of SARS-CoV-2 in the sample. The test is based on reverse transcribing RNA to cDNA and subsequent amplification with an RT-qPCR instrument. Designed primers are targeting different parts of the SARS-CoV-2 genetic sequence, of which, gene E (Envelope) is believed to be of the highest sensitivity (D’Cruz et al., 2020).

Every RT-qPCR assay needs to be thoroughly validated before it is used in a clinical setting. Validation should be done for each laboratory based on the RT-qPCR methodology that you use. The objective is to determine the maximum number of pooled samples that do not affect the method’s accuracy. ECDC’s suggestion of a maximum number of pooled samples is 32 per pool, but this number may greatly vary on the RT-qPCR protocols that a laboratory uses and the limit of detection of the RT-qPCR assay (ECDC, 2020). More on validation studies can be read here.

RT-qPCR challenges

While RT-qPCR is a method that gets us the best results in relation to time efficiency and reliability, there are still some challenges that it faces:

False-positive test results

False-positive test results can occur due to different reasons.

1. A cross-reaction from primers and nucleic acids originating in co-infection with different viruses or bacteria. To avoid this kind of mistake, matching of SARS-CoV-2 primer and probes needs to be done with the use of reliable libraries.

2. Contamination of laboratory reagents: Another cause for false-positive test results can be contamination of laboratory reagents, which can happen more often with a higher volume of tests done. The contamination occurs on a microtiter plate itself when positive sample content is accidentally transmitted to the well(s) near to it either through a contaminated pipetting tip or by unintentional shaking of the plate. One can also get false-positive results if reagents are contaminated due to inappropriate handling. For example, if you are pipetting reagents and samples in the same hood or pipetting reagents with contaminated tips. In this case, the wrong interpretation of results can be avoided by using negative controls (D’Cruz et al., 2020).

False-negative test results

False-negative results of COVID-19 diagnostics can be a consequence of the sensitivity of COVID-19 RT-qPCR tests or the inaccuracies occurring during the actual execution of RT-qPCR.

1. Sensitivity of an RT-qPCR test. The sensitivity of RT-qPCR COVID-19 tests is reported to be 90% (Colin P. et al., 2020), which means that if a country has 5 million infected individuals, 500 000 would be falsely classified as free of infection. A consecutive testing (i.e. 2 tests done in a range of a few days) might help set the right diagnosis.

2. Pipetting mistakes: A false negative result can be a consequence of a scenario where a technician would:

  • Accidentally miss the well, which would not contain a potentially positive sample.
  • Fail to add all the necessary reagents.

3. Poor reagent quality: Sometimes reagents may not be working properly due to poor design or inappropriate storage. In that case, you would observe negative results on your positive control samples. To validate negative results, tests should be performed with a different primer or reagent set.

4. Mutations in a viral genome: In a very unlikely scenario, false-negative test results can occur due to mutations in the primer and/or probe target regions in the SARS-CoV-2 genome. To validate negative results, the test should be performed again with different primer sets against the same gene target. Outcomes should be joined with the patient’s history and other clinical data, to determine accurate patient infection status (D’Cruz et al., 2020).

Materials supply scarcity

Due to high interest and the need for RT-qPCR tests, a lack of supply occurred in the first wave of the COVID-19 pandemic. To overcome this challenge, different reference panels and RT-qPCR protocols are established to release the pressure of a single manufacturer to produce all the required materials.

RT-qPCR protocols

With time, there are more and more non-commercial (so-called in-house tests) and commercial (in vitro diagnostic medical devices or IVDs) SARS-CoV-2 tests emerging. What best suits your laboratory depends on your location and current policies, laboratory equipment and staff number, and education. WHO prepared a summary table of some common in-house protocols. Moreover WHO Collaborating Centre for Laboratory Strengthening and Diagnostic Technology Evaluation established a searchable database FIND where commercial SARS-CoV-2 test is available to find in one place.

CDC produced two in-house protocols for RT-qPCR tests. Both protocols are approved by the FDA and were granted EUA (Emergency Use Authorization). Reagents can be ordered through IRR (International Reagent Resource)

FDA also granted an amendment to CDC with approved possible alternatives in testing materials, comprised of additional extraction reagents, additional extraction instruments, and associated reagents and new processes that can be used in place of the extraction methods when materials for the current method are limited. You can view the document with alternatives here.

ECDC built COVID-19 In Vitro Diagnostic Devices and Test Methods Database where you can search for different IVDs or methods and also determine if it can be sold within the European Economic Area.

Institut Pasteur, Paris produced RT-qPCR protocol for the detection of SARS-CoV-2 for two RdRp targets (IP2 and IP4).

Preanalytical and analytical errors

RT-qPCR is currently the gold standard for identifying SARS-CoV-2 infection and for that reason, it is of great importance that we account for potential vulnerabilities of the method. Lippi et al. (2020) emphasized the possibilities of different preanalytical and analytical issues, that we have also mentioned above.  These include inadequate procedures of collecting samples and identification, manual errors, sample contamination, active viral recombination, use of invalid assays, etc. With increasing numbers of tests, there is also increased pressure on laboratory staff that can make more manual mistakes due to stress and unusually big workload.

That is where BioSistemika can help to fight against the COVID-19 pandemic. We developed Pipetting Aid PlatR, an easy to use tablet-based application, that illuminates the wells on your microtiter plate. You can import your samples and use a pre-defined COVID-19 protocol. PlatR already helps more than 150 laboratories worldwide, many of which are COVID-testing diagnostic laboratories, to produce reliable results, minimize errors, and increase throughput. The use of PlatR will increase your productivity and enable you to make fewer pipetting mistakes, as our research showed that you can get up to 26,7% faster with pipetting while making zero mistakes.

To further show our dedication to be a part of the solution to the COVID-19 crisis, we will import your most commonly used COVID-19 protocols to PlatR, so that you can start using it immediately without any need to train your staff.

For more information about PlatR, please visit our website.