Analytical sensitivity and efficiency comparisons of SARS-COV-2 qRT-PCR assays

The recent spread of severe acute respiratory syndrome coronavirus (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt public health actions. Currently, several quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assays are being used by clinical, research, and public health laboratories for rapid detection of the virus. However, it is currently unclear if results from different tests are comparable. Our goal was to evaluate the primer-probe sets used in four common diagnostic assays available on the World Health Organization (WHO) website. To facilitate this effort, we generated RNA transcripts to create standards and distributed them to other laboratories for internal validation. We then used these RNA transcript standards, full-length SARS-CoV-2 RNA, and RNA-spiked mock samples to determine analytical efficiency and sensitivity of nine primer-probe sets. We show that all primer-probe sets can be used to detect SARS-CoV-2, but there are clear differences in the ability to differentiate between true negatives and positives with low amounts of virus. Adding to this, many primer-probe sets, including the "N2" and "N3" sets issued by the US Centers for Disease Control and Prevention, have background amplification with SARS-CoV-2-negative nasopharyngeal swabs, which may lead to inconclusive results. Our findings characterize the limitations of commonly used primer-probe sets and can assist other laboratories in selecting appropriate assays for the detection of SARS-CoV-2.