US CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome Coronavirus 2

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the etiologic agent associated with coronavirus disease, which emerged in late 2019. In response, we developed a diagnostic panel consisting of 3 real-time reverse transcription PCR assays targeting the nucleocapsid gene and evaluated use of these assays for detecting SARS-CoV-2 infection. All assays demonstrated a linear dynamic range of 8 orders of magnitude and an analytical limit of detection of 5 copies/reaction of quantified RNA transcripts and 1 x 10^-1.5 50% tissue culture infectious dose/mL of cell-cultured SARS-CoV-2. All assays performed comparably with nasopharyngeal and oropharyngeal secretions, serum, and fecal specimens spiked with cultured virus. We obtained no false-positive amplifications with other human coronaviruses or common respiratory pathogens. Results from all 3 assays were highly correlated during clinical specimen testing. On February 4, 2020, the Food and Drug Administration issued an Emergency Use Authorization to enable emergency use of this panel.

Keywords: 2019 novel coronavirus disease; COVID-19; SARS-CoV-2; coronavirus disease; real-time RT-PCR; real-time reverse transcription PCR; respiratory infections; severe acute respiratory syndrome coronavirus 2; viruses; zoonoses.

Figure 1

Linear regression analysis of serial 10-fold dilutions of synthetic RNA transcripts of the nucleocapsid gene (N) ranging from 5 to 5 × 10⁷ copies/reaction tested by the N1 (A), N2 (B), and N3 (C) assays in the US Centers for Disease Control and Prevention real-time reverse transcription PCR panel for detection of severe acute respiratory syndrome coronavirus 2. For each assay, R² indicates calculated linear correlation coefficients and eff. indicates amplification efficiencies. C_t, cycle threshold.

Figure 2

Comparison of the N1, N2, and N3 assays in the US Centers for Disease Control and Prevention real-time reverse transcription PCR panel for detection of SARS-CoV-2 with 223 SARS-CoV-2–positive clinical specimens. Linear regression lines were fitted to C_t values, with regression equations and coefficients of determination (R²). A) N1 vs. N2; B) N1 vs. N3; C) N2 vs. N3. C_t, cycle threshold; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.