How to choose the right real-time RT-PCR primer sets for the SARS-CoV-2 genome detection?

Abstract The SARS-CoV-2 pandemic has created an unprecedented need for rapid large-scale diagnostic testing. Currently, several quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assays recommended by the World Health Organization are being used by clinical and public health laboratories and typically target regions of the RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N) coding region. However, it is currently unclear if results from different tests are comparable. The present study demonstrates substantial differences in SARS-CoV-2 RNA detection sensitivity among the primer/probe sets recommended by the World Health Organization especially for low-level viral loads. The alignment of thousands of European SARS-CoV-2 sequences against the primers/probe highlights single mismatches which might also contribute to false negatives. An understanding of the limitations depending on the targeted genes and primer/probe sets may influence the selection of molecular detection assays by clinical laboratories.