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Assessing qRT-PCR COVID-19 Testing Results: What Can Go Wrong?

Sep 20, 2021

Nisreen Shumayrikh, PhD
© Liszewski/Qiagen

COVID-19 is commonly diagnosed by quantitative fluorescence real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) (1, 2). The test is the sequential action of two enzymes--an RNA-dependent DNA polymerase, also known as a reverse transcriptase, to copy the SARS-CoV-2 RNA into complementary DNA (cDNA), and Taq polymerase to amplify the cDNA by traditional PCR. 

In addition, the protocol requires primers—short single-stranded DNA fragments complementary to regions flanking the part of the viral cDNA sequence to be amplified. To detect the amplified viral DNA, scientists modify these primers with fluorescence labels. 

The mixture is then placed into the qRT-PCR thermal cycler, which cycles through heating and cooling processes to trigger separation and annealing of DNA, creating new, identical copies of the target sections of viral DNA. A standard real-time qRT-PCR setup usually goes through 30 - 40 cycles, generating millions of copies of the viral DNA from each strand present in the sample. 

Although the test’s sensitivity and specificity, which determine overall accuracy, are considerably high, scientists question its reliability. The high sensitivity increases the chance of detecting any RNA present in the sample, leading to false-positive results. On the other hand, false-negative findings can arise if the viral RNA load is low or primers fail to anneal to the viral sequence. 

An article published by Mayo Clinic Proceedings sheds light on the risk of trusting COVID-19 testing results to make clinical and public health decisions (3). Priya Sampathkumar, MD, an infectious diseases specialist at Mayo Clinic and a co-author, says: “RT-PCR testing is most useful when it is positive. It is less useful in ruling out COVID-19. A negative test often does not mean the person does not have the disease, and test results need to be considered in the context of patient characteristics and exposure” (4).

The article focuses on three crucial considerations regarding assessing qRT-PCR results.

Varying viral RNA sequences

Scientists have documented SARS-CoV-2’s genetic diversity and rapid evolution in several studies (5, 6). Different qRT-PCR kits are now commercially available but their specificity and sensitivity vary, mainly because of the primers’ design. Mutations in the SARS-CoV-2 genome can cause mismatches between the primer and its target sequence, increasing the number of possible false-negative results. 

To overcome this challenge, repeat testing using a different kit with primers designed to amplify other, non-overlapping regions of the viral DNA may be helpful. Additionally, the clinical conditions and the history of exposure can aid in interpreting the results, especially if the data are not conclusive. Furthermore, results obtained from various tests, instruments, laboratories, and countries, if documented and compared, can help develop standardized guidelines for optimally detecting SARS-CoV-2 through qRT-PCR. 

Threshold considerations

The cycle threshold value (Ct) is the minimum number of PCR cycles needed for the qRT-PCR test to successfully detect the virus. It indicates an estimate, not the exact amount, of the viral DNA present in the sample. If the viral DNA is detected at a low cycle number (Ct value under 30), it means that the virus is easier to detect, and a large amount of the viral genetic material is present in the starting sample. 

Ct values are influenced by factors such as the test kit, the viral load in the sample collected, the machine used for testing, the technique used to collect the sample, and the sample type. Since Ct values are not standardized within provinces or countries, they are not directly comparable, which can add to the complexity of interpreting the reliability of the COVID-19 test.

Sample collection and transport 

Proper biospecimen collection is a vital step in the diagnosis of infectious diseases. In the case of SARS-CoV-2, the nasopharyngeal swab is the reference sampling method as recommended by the World Health Organization (7). Once the technician collects the samples, these should be transported immediately to the testing laboratory, maintaining the samples at cold temperatures of 4°C or lower during transport. In case of delays, the specimen can be stored at 2 - 8 °C for up to 72 hours after collection. 

If further delays are expected, samples should be stored at -20°C or ideally at -70 °C freezers and shipped on dry ice. It is essential to avoid repeated freezing and thawing cycles of specimens as that increases the chances of fragmenting genetic material.

Extracted RNA should be stored at -20°C for retesting if required. Failing to follow these instructions can result in degrading the isolated viral RNA which in turn results in false-negative or inconclusive results with increased Ct values. 

As the SARS-CoV-2 pandemic evolves, the reliability and accuracy of qRT-PCR tests are crucial for controlling the spread of the virus and determining public health measures. 

References

  1. Bustin SA, Nolan T., RT-qPCR Testing of SARS-CoV-2: A Primer. International Journal of Molecular Sciences. 2020;21(8):3004. https://doi.org/10.3390/ijms21083004
  2. Carter LJ, Garner LV, Smoot JW, Li Y, Zhou Q, Saveson CJ, Sasso JM, Gregg AC, Soares DJ, Beskid TR, Jervey SR, Liu C. Assay techniques and test development for COVID-19 diagnosis. ACS Cent Sci. 2020;6(5):591–605. https://doi.org/10.1021/acscentsci.0c00501
  3. Colin P. West, Victor M. Montori, Priya Sampathkumar. COVID-19 Testing: The Threat of False-Negative Results. Mayo Clinic Proceedings. 2020;95(6):1127- 1129. https://doi.org/10.1016/j.mayocp.2020.04.004
  4. Mayo Clinic. (2020, April 9). False-negative COVID-19 test results may lead to false sense of security. ScienceDaily. Retrieved September 17, 2021 from www.sciencedaily.com/releases/2020/04/200409144805.htm
  5.  Phan T. Genetic diversity and evolution of SARS-CoV-2. Infect Genet Evol. 2020;81:104260. https://doi.org/10.1016/j.meegid.2020.104260
  6. Shen Z, Xiao Y, Kang L, Ma W, Shi L, Zhang L, Zhou Z, Yang J, Zhong J, Yang D, Guo L, Zhang G, Li H, Xu Y, Chen M, Gao Z, Wang J, Ren L, Li M. Genomic diversity of severe acute respiratory syndrome-coronavirus 2 in patients with coronavirus disease 2019. Clin Infect Dis. 2020;71(15):713–720. https://doi.org/10.1093/cid/ciaa203
  7. World Health Organization (WHO). Laboratory testing for coronavirus disease (COVID-19) in suspected human cases Interim guidance 19 March 2020. https://www.who.int/publications/i/item/10665-331501 

 

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