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

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.

Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

“…To control for inhibitors of the reverse transcriptase or PCR enzymes and/or RNase activity in the swab diluent, an aliquot of each swab diluent was spiked with 4 × 10 4 copies of EXO control RNA for subsequent detection with an EXO primer/probe set. While it has been reported that the CDC RNase P primers have the potential to amplify both genomic DNA as well as mRNA (as both primers are in the same exon), the EXO spike-in control utilized in the UW LDT provides a built-in control for successful RT-PCR amplification from a known RNA template [ 9 ]. Inhibition was noted in only 2 samples of 240 tested, 1 negative sample and 1 positive sample tested by the direct approach ( S1B and S2B Tables).…”

A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection

“…Both M and S gene assays have been shown to have high sensitivity [20,21], and their target regions display low sequence variation (S5 Table). Additional improvements to our protocol could include the use of control primers/probe specific to a human RNA transcript (the RPP30 primers/probe described here detect both RNA and genomic DNA) as this would ensure that samples contain intact RNA [22]. However, it should be stressed that any changes to the protocol may also change the sensitivity of SARS-CoV-2 detection, and new protocols should undergo appropriate validation before use for diagnostic purposes.…”

A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection

“…Both M and S gene assays have been shown to have high sensitivity [20, 21] and their target regions display low sequence variation (Table S5). Additional improvements to our protocol could include the use of control primers/probe specific to a human RNA transcript (the RPP30 primers/probe described here detect both RNA and genomic DNA) as this would ensure that samples contain intact RNA [22]. However, it should be stressed that any changes to the protocol may also change the sensitivity of SARS-CoV-2 detection, and new protocols should undergo appropriate validation before use for diagnostic purposes.…”