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Overhauling a faulty control in the CDC-recommended SARS-CoV-2 RT-PCR test panel

Rob J. Dekker, Wim A. Ensink, Selina van Leeuwen, Han Rauwerda, Timo M. Breit
doi: https://doi.org/10.1101/2020.06.12.147819
Rob J. Dekker
1RNA Biology & Applied Bioinformatics research group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam (UvA), Amsterdam 1098 XH, the Netherlands
2MAD: Dutch Genomics Service & Support Provider, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam (UvA), Amsterdam 1098 XH, the Netherlands
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Wim A. Ensink
1RNA Biology & Applied Bioinformatics research group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam (UvA), Amsterdam 1098 XH, the Netherlands
2MAD: Dutch Genomics Service & Support Provider, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam (UvA), Amsterdam 1098 XH, the Netherlands
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Selina van Leeuwen
1RNA Biology & Applied Bioinformatics research group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam (UvA), Amsterdam 1098 XH, the Netherlands
2MAD: Dutch Genomics Service & Support Provider, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam (UvA), Amsterdam 1098 XH, the Netherlands
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Han Rauwerda
1RNA Biology & Applied Bioinformatics research group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam (UvA), Amsterdam 1098 XH, the Netherlands
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Timo M. Breit
1RNA Biology & Applied Bioinformatics research group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam (UvA), Amsterdam 1098 XH, the Netherlands
2MAD: Dutch Genomics Service & Support Provider, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam (UvA), Amsterdam 1098 XH, the Netherlands
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  • For correspondence: t.m.breit@uva.nl
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ABSTRACT

To battle the COVID-19 pandemic, widespread testing for the presence of the SARS-CoV-2 virus is worldwide being employed by specific real-time RT-PCR (rRT-PCR) of viral RNA. The CDC has issued a recommended panel of PCR-based test sets that entail several primer/probe sets that target the SARS-CoV-2 N-gene, but also one that targets the human RNase P gene (h-RP) as a positive control for RNA extraction and/or reverse-transcription (RT) efficacy.

We discovered that the CDC-recommended h-RP primer/probe set has a faulty design, because both PCR primers are located in the same exon, which allows for unwanted PCR-amplification of background genomic DNA (gDNA). By removing RNA from nose-swab samples by an RNase treatment, we showed that the presence of gDNA in samples resulted in false-positive signals for the h-RP test control. This is rather serious, because it could lead to false-negative test outcomes, since the CDC interpretation of an absent SARS-CoV-2 rRT-PCR signal plus a positive h-RP rRT-PCR signal is interpreted as “2019-nCoV not detected”, whereas a false-positive h-RP rRT-PCR signal resulting from amplification of gDNA should be interpreted as “Invalid Result” and the procedure should be repeated.

In order to overhaul the faulty h-RP rRT-PCR primer/probe set with minimal modification, we designed and tested several new h-RP reverse primers. Replacement of the CDC-recommended PCR reverse primer with our selected exon-exon junction reverse primer corrected the problem of false-positive results with this important SARS-CoV-2 RT-PCR test control and thus eliminated the problem of potential false-negative COVID-19 diagnoses.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Relevant study discovered during submission, small paragraph concerning this study was added in the concluding remarks and the related reference added to the references.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 17, 2020.
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Overhauling a faulty control in the CDC-recommended SARS-CoV-2 RT-PCR test panel
Rob J. Dekker, Wim A. Ensink, Selina van Leeuwen, Han Rauwerda, Timo M. Breit
bioRxiv 2020.06.12.147819; doi: https://doi.org/10.1101/2020.06.12.147819
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Overhauling a faulty control in the CDC-recommended SARS-CoV-2 RT-PCR test panel
Rob J. Dekker, Wim A. Ensink, Selina van Leeuwen, Han Rauwerda, Timo M. Breit
bioRxiv 2020.06.12.147819; doi: https://doi.org/10.1101/2020.06.12.147819

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