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Review
. 2022 Feb;22(2):157-167.
doi: 10.1080/14737159.2022.2037425. Epub 2022 Feb 21.

True or false: what are the factors that influence COVID-19 diagnosis by RT-qPCR?

Luina Benevides Lima  1 , Felipe Pantoja Mesquita  1 , Lais Lacerda Brasil de Oliveira  1 , Francisca Andréa da Silva Oliveira  1 , Maria Elisabete Amaral de Moraes  1 , Pedro F N Souza  2 , Raquel Carvalho Montenegro  1
Affiliations
Review

True or false: what are the factors that influence COVID-19 diagnosis by RT-qPCR?

Luina Benevides Lima et al. Expert Rev Mol Diagn. 2022 Feb.

Abstract

Introduction: The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease has had a catastrophic impact on the world resulting in several deaths. Since World Health Organization declared the pandemic status of the disease, several molecular diagnostic kits have been developed to help the tracking of viruses spread.

Areas covered: This review aims to describe and evaluate the currently reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) diagnosis kit. Several processes used in COVID-19 diagnostic procedures are detailed in further depth to demonstrate optimal practices. Therefore, we debate the main factors that influence the viral detection of SARS-COV-2 and how they can affect the diagnosis of patients.

Expert opinion: Here is highlighted and discussed several factors that can interfere in the RT-PCR analysis, such as the viral load of the sample, collection site, collection methodology, sample storage, transport, primer, and probe mismatch/dimerization in different brand kits. This is a pioneer study to discuss the factor that could lead to the wrong interpretation of RT-qPCR diagnosis of SARS-CoV-2. This study aimed to help the readers to understand what very likely is behind a bad result of SARS-CoV-2 detection by RT-PCR and what could be done to reach a reliable diagnosis.

Keywords: COVID-19; Coronavirus; RT-PCR; diagnosis; sensitivity.

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Figures

Figure 1.
Figure 1.
Morphology of the three relevant coronaviruses with epidemic/pandemic impact on human health. SARS-CoV-1, MERS-CoV and SARS-CoV-2. The Structural proteins are shown in the figure, such as spike (S), membrane (M), nucleocapsid (N) and envelope (E). Their genes are commonly used as target for real time PCR detection. Created in BioRender.com.
Figure 2.
Figure 2.
Scheme showing gene amplification in RT-qPCR process. In the RT-qPCR the first step is the construction of complementary DNA (cDNA) using RNA as model by reverse transcriptase. Thereafter, the stable double-strand DNA is used as template for the exponentially amplification of the product. Created in BioRender.com.
Figure 3.
Figure 3.
Flow Chart for COVID-19 diagnostic by RT-qPCR. Patients presenting symptoms of COVID-19 are subjected to the test. (1) occurs the sample collection by either naso- or oropharyngeal. (2) The collected sample is immediately processed. (3) RNA extraction. (4) RT-qPCR process and (5) Data analysis. Created in BioRender.com.
Figure 4.
Figure 4.
Flow Chart suggesting the best time to perform COVID-19 diagnosis. After exposition to SARS-CoV-2, symptomatic stage is going until 2 weeks after contact, which is the best time to SARS-CoV-2 detection. In normal patients and compromised patients’ low viral loads could still be detected in 3–4 weeks. Created in BioRender.com.
See this image and copyright information in PMC

References

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