Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets

Abstract

The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse transcription-PCR (RT-qPCR) assays are being used by clinical, research and public health laboratories. However, it is currently unclear whether results from different tests are comparable. Our goal was to make independent evaluations of primer-probe sets used in four common SARS-CoV-2 diagnostic assays. From our comparisons of RT-qPCR analytical efficiency and sensitivity, we show that all primer-probe sets can be used to detect SARS-CoV-2 at 500 viral RNA copies per reaction. The exception for this is the RdRp-SARSr (Charité) confirmatory primer-probe set which has low sensitivity, probably due to a mismatch to circulating SARS-CoV-2 in the reverse primer. We did not find evidence for background amplification with pre-COVID-19 samples or recent SARS-CoV-2 evolution decreasing sensitivity. Our recommendation for SARS-CoV-2 diagnostic testing is to select an assay with high sensitivity and that is regionally used, to ease comparability between outcomes.

Extended Data Fig. 1. Generation of RNA transcript standards for validation of SARS-CoV-2 RT-qPCR assays.

a, SARS-CoV-2 genome locations of generated RNA transcript standards for the non-structural protein 10 (nsp10), RNA-dependent RNA polymerase (RdRp), non-structural protein 14 (nsp14), envelope (E), and nucleocapsid (N) genes and the nine primer-probe sets used RT-qPCR assays. b, The slope, intercept, R², and efficiency of RT-qPCR using tenfold dilutions (10⁰-10⁶ viral RNA copies/μL) of RNA transcript standards with the corresponding primer-probe sets. Shown are mean Ct values based on 2 technical replicates. The primer-probe sets are numbered as shown in panel A. The RNA transcript primers and sequences can be found in Supplementary Table 2 and Supplementary Table 3, respectively. Data used to make this figure can be found in Source Data Extended Data Fig. 1. Source data

Extended Data Fig. 2. No effect of different concentrations of RdRp-SARSr primers and probes on analytical sensitivity.

Low performance of the standardized RdRp-SARSr primer-probe set triggered us to further investigate the effect of primer concentrations. We compared our standardized primer-probe concentrations (500 nM of forward and reverse primers, and 250 nM of probe) with the recommended concentrations in the confirmatory assay (600 nM of forward primer, 800 nM of reverse primer, 100 nM of probe 1, and 100 nM of probe 2), and the discriminatory assay (600 nM of forward primer, 800 nM of reverse primer, and 200 nM of probe 2) as developed by the Charité Institute of Virology Universitätsmedizin Berlin. Standard curves for both RdRp-transcript standard and full-length SARS-CoV-2 RNA are similar, which indicates that higher primer concentrations did not improve the performance of the RdRp-SARSr set. Symbol indicates tested sample type (circles = RdRp transcript standard, and squares = full-length SARS-CoV-2 RNA from cell culture) and colors indicate the different primer and probe concentrations. Data used to make this figure can be found in Source Data Extended Data Fig 2. Source data

Fig. 1. Analytical efficiency and sensitivity of the nine primer-probe sets used in SARS-CoV-2 qRT-qPCR assays.

a,b, Mean Ct values for nine primer-probe sets and a human control primer-probe set targeting the human RNase P gene tested for 2 technical replicates with 10-fold dilutions of (a) full-length SARS-CoV-2 RNA and (b) pre-COVID-19 nasopharyngeal swabs spiked with known concentrations of SARS-CoV-2 RNA (SARS-CoV-2 RNA-spiked mocks). The CDC human RNase P (RP) assay was included as an extraction control. c,d, From the dilution curves in panels a and b, (c) PCR efficiency and (d) y-intercept Ct values (measured analytical sensitivity) were calculated for each of nine primer-probe sets. Symbols depict sample types: squares represent tests with SARS-CoV-2 RNA and diamonds represent SARS-CoV-2 RNA-spiked mock samples. Colors depict the nine tested primer-probe sets. The primer and probe sequences can be found in Supplementary Table 1. Data used to make this figure can be found in Source Data Fig. 1.

Fig. 2. Comparison of analytical sensitivity of SARS-CoV-2 primer-probe sets using pre-COVID-19 nasopharyngeal swabs.

The lower detection limit of nine primer-probe sets as well as the human RNase P control from RNA extracted from nasopharyngeal swabs collected in 2017 spiked with known concentrations of SARS-CoV-2 RNA. Each primer-probe set was performed using 24 technical replicates of pooled swab RNA without spiking SARS-CoV-2 RNA (‘no virus’; 6 replicates with 4 independent pools of 4 swabs) and 8 replicates (2 replicates with 4 independent pools of each 4 nasopharyngeal swabs) spiked with 10⁰–10² viral RNA copies/μL of SARS-CoV-2 RNA. ND = not detected. Black lines indicate the median and the dashed line indicates the detection limit. Data used to make this figure can be found in Source Data Fig. 2.

Fig. 3. Low rate of inconclusive testing outcomes using the US CDC N1 and N2 primer-probe sets.

Clinical samples negative or low positive for SARS-CoV-2 were used to determine if differences between the analytical sensitivities of the US CDC N1 and N2 primers produced inconclusive results. a, Cycle threshold (Ct) values for the same 172 clinical samples testing using the N1 and N2 primer probe sets. b, We compared Ct values obtained with the two primer-probe sets for clinical samples with Ct values higher than 35. N1 = 2019-nCoV_N1, N2 = 2019-nCoV_N2, ND = not detected. Solid black line indicates the median, and dashed line indicates the detection limit. Data used to make this figure can be found in Source Data Fig. 3.

Fig. 4. Genetic diversity of available SARS-CoV-2 genomes.

992 SARS-CoV-2 genomes available as of 22 March 2020 (listed in Source Data Fig. 4) were aligned to calculate nucleotide diversity and investigate mismatches with the nine primer-probe sets. Genetic diversity was measured using pairwise identity (%) at each position, disregarding gaps and ambiguous nucleotides. Asterisks (*) at the top indicate primers (green) and probes (red) targeting regions with one or more mismatches. Genomic plots were designed using DNA Features Viewer 3.0.1 in Python version 3.7.