In vitro evaluation of the effect of mutations in primer binding sites on detection of SARS-CoV-2 by RT-qPCR

Zimmermann, Fee; Urban, Maria Letizia; Krüger, Christian; Walter, Mathias C.; Wölfel, Roman; Zwirglmaier, Katrin

doi:10.1016/j.jviromet.2021.114352

Cited by 15 publications

(18 citation statements)

References 21 publications

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“…On the other hand, the number of samples that had damaged TRs for more than half of the primer systems in the set (samples "susceptible to misclassi cation") was negligible for all investigated primer sets. This underlines the importance of using more than one target in diagnostic PCR tests already pointed out by previous studies 28, 34 .…”

Section: Discussionsupporting

confidence: 75%

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Identification of mutations in SARS-CoV-2 PCR primer regions

Mentes

Papp

Visontai

et al. 2022

Preprint

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Due to the constantly increasing number of mutations in the SARS-CoV-2 genome, concerns have emerged over the possibility of decreased diagnostic accuracy of reverse transcription-polymerase chain reaction (RT-PCR), the gold standard diagnostic test for SARS-CoV-2. We propose an analysis pipeline to discover genomic variations overlapping the target regions of commonly used PCR primer sets. We provide the list of these mutations in a publicly available format based on a dataset of more than 600,000 SARS-CoV-2 samples. Our approach distinguishes among mutations possibly having a damaging impact on PCR efficiency and ones anticipated to be neutral in this sense. Samples are categorized as „prone to misclassification” vs. „likely to be correctly detected” by a given PCR primer set based on the estimated effect of mutations present. Samples susceptible to misclassification are always present at a daily rate of 2% or lower, while the daily ratio of samples having a slight chance of misclassification with a particular primer set can reach 90%. As different variant strains may temporarily gain dominance in the worldwide SARS-CoV-2 viral population, the efficiency of a particular PCR primer set may change over time, therefore constant monitoring of variations in primer target regions is highly recommended.

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Section: Discussionsupporting

confidence: 75%

“…Zimmermann et al 34 highlighted the fact that experimental data does not necessarily follow the theoretical predictions, particularly with regard to the magnitude of the Ct shift with mismatches close to the 3' end. Moreover, the speci c nucleotide composition of these mismatches also seemed to play a role in determining PCR e cacy 35 .…”

Section: Discussionmentioning

confidence: 99%

Identification of mutations in SARS-CoV-2 PCR primer regions

Mentes

Papp

Visontai

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Zimmermann et al 34 highlighted the fact that experimental data does not necessarily follow the theoretical predictions, particularly with regard to the magnitude of the Ct shift with mismatches close to the 3′ end. Moreover, the specific nucleotide composition of these mismatches also seemed to play a role in determining PCR efficacy 35 .…”

Section: Discussionmentioning

confidence: 99%

Identification of mutations in SARS-CoV-2 PCR primer regions

Mentes

Papp

Visontai

et al. 2022

Sci Rep

View full text Add to dashboard Cite

Due to the constantly increasing number of mutations in the SARS-CoV-2 genome, concerns have emerged over the possibility of decreased diagnostic accuracy of reverse transcription-polymerase chain reaction (RT-PCR), the gold standard diagnostic test for SARS-CoV-2. We propose an analysis pipeline to discover genomic variations overlapping the target regions of commonly used PCR primer sets. We provide the list of these mutations in a publicly available format based on a dataset of more than 1.2 million SARS-CoV-2 samples. Our approach distinguishes among mutations possibly having a damaging impact on PCR efficiency and ones anticipated to be neutral in this sense. Samples are categorized as “prone to misclassification” vs. “likely to be correctly detected” by a given PCR primer set based on the estimated effect of mutations present. Samples susceptible to misclassification are generally present at a daily rate of 2% or lower, although particular primer sets seem to have compromised performance when detecting Omicron samples. As different variant strains may temporarily gain dominance in the worldwide SARS-CoV-2 viral population, the efficiency of a particular PCR primer set may change over time, therefore constant monitoring of variations in primer target regions is highly recommended.

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“…However, the proportion of discordant double tests registered in the 4th period (76%) is percentage-wise larger than the proportion of double positive test results in the period (51.2%), which may indicate that the increasing incidence of COVID-19 in Denmark during the 4th period cannot explain this finding alone. Additionally, it should be considered that mutations of the SARS-CoV-2 virus are developing during the pandemic, thus affecting the sensitivity and specificity of the RT-PCR analysis over time [ 9 , 10 , 11 , 12 , 13 ]. In Denmark, the original SARS-CoV-2 virus strain was dominating during most of our study period (1st March 2020–1st March, 2021), however the alpha-subtype of the virus strain was registered in November 2020, and it became the dominating SARS-CoV-2 strain in March 2021 [ 14 ].…”

Section: Discussionmentioning

confidence: 99%

A Comparison of Pharyngeal Swabs and Tracheal Secretions for the Diagnosing of COVID-19

et al. 2022

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The aim of this study was to compare the test results from patients who, within a short timescale, have been tested for COVID-19 using both a pharyngeal swab and tracheal secretion. Data were collected from the database of AUH, from patients hospitalized between 1 March 2020 and 1 March 2021 who, due to symptoms of COVID-19, were tested by a pharyngeal swab and by tracheal secretion. We found great agreement between oropharyngeal swab and tracheal secretion RT-PCR testing for the diagnosis of COVID-19, with 98.5% of double tests being concordant and only 1.5% being discordant. This finding may advocate a single-test strategy being either an oropharyngeal swab RT-PCR testing or tracheal secretion, although this study revealed 15.9% false negative oropharyngeal swabs.

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In vitro evaluation of the effect of mutations in primer binding sites on detection of SARS-CoV-2 by RT-qPCR

Cited by 15 publications

References 21 publications

Identification of mutations in SARS-CoV-2 PCR primer regions

Identification of mutations in SARS-CoV-2 PCR primer regions

Identification of mutations in SARS-CoV-2 PCR primer regions

A Comparison of Pharyngeal Swabs and Tracheal Secretions for the Diagnosing of COVID-19

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