Patient DNA cross-reactivity of the CDC SARS-CoV-2 extraction control leads to an inherent potential for false negative results

Testing for RNA viruses such as SARS-CoV-2 requires careful handling of inherently labile RNA during sample collection, clinical processing, and molecular analysis. Tests must include fail-safe controls that affirmatively report the presence of intact RNA and demonstrate success of all steps of the assay. A result of “no virus signal” is insufficient for clinical interpretation: controls must also say “The reaction worked as intended and would have found virus if present.” Unfortunately, a widely used test specified by the US Centers for Disease Control and Prevention (CDC) incorporates a control that does not perform as intended and claimed. Detecting SARS-CoV-2 with this assay requires both intact RNA and successful reverse transcription. The CDC-specified control does not require either of these, due to its inability to differentiate human genomic DNA from reverse-transcribed RNA. Patient DNA is copurified from nasopharyngeal swabs during clinically-approved RNA extraction and is sufficient to return an “extraction control success” signal using the CDC design. As such, this assay fails-unsafe: truly positive patient samples return a false-negative result of “no virus detected, control succeeded” following any of several readily-encountered mishaps. This problem affects tens-of-millions of patients worth of shipped assays, but many of these flawed reagents have not yet been used. There is an opportunity to improve this important diagnostic tool. As demonstrated here, a re-designed transcript-specific control correctly monitors sample collection, extraction, reverse transcription, and qPCR detection. This approach can be rapidly implemented and will help reduce truly positive patients from being incorrectly given the all-clear.