PT  - JOURNAL ARTICLE
AU  - Rob J. Dekker
AU  - Wim A. Ensink
AU  - Selina van Leeuwen
AU  - Han Rauwerda
AU  - Timo M. Breit
TI  - Overhauling a faulty control in the CDC-recommended SARS-CoV-2 RT-PCR test panel
AID  - 10.1101/2020.06.12.147819
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.06.12.147819
4099  - http://biorxiv.org/content/early/2020/06/17/2020.06.12.147819.short
4100  - http://biorxiv.org/content/early/2020/06/17/2020.06.12.147819.full
AB  - 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 StatementThe authors have declared no competing interest.