Analytical comparisons of SARS-COV-2 detection by qRT-PCR and ddPCR with multiple primer/probe sets

Abstract

Different primers/probes sets have been developed all over the world for the nucleic acid detection of SARS-CoV-2 by quantitative real time polymerase chain reaction (qRT-PCR) as a standard method. In our recent study, we explored the feasibility of droplet digital PCR (ddPCR) for clinical SARS-CoV-2 nucleic acid detection compared with qRT-PCR using the same primer/probe sets issued by Chinese Center for Disease Control and Prevention (CDC) targeting viral ORF1ab or N gene, which showed that ddPCR could largely minimize the false negatives reports resulted by qRT-PCR [Suo T, Liu X, Feng J, et al. ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens. medRxiv [Internet]. 2020;2020.02.29.20029439. Available from: https://medrxiv.org/content/early/2020/03/06/2020.02.29.20029439.abstract]. Here, we further stringently compared the performance of qRT-PCR and ddPCR for 8 primer/probe sets with the same clinical samples and conditions. Results showed that none of 8 primer/probe sets used in qRT-PCR could significantly distinguish true negatives and positives with low viral load (10^-4 dilution). Moreover, false positive reports of qRT-PCR with UCDC-N1, N2 and CCDC-N primers/probes sets were observed. In contrast, ddPCR showed significantly better performance in general for low viral load samples compared to qRT-PCR. Remarkably, the background readouts of ddPCR are relatively lower, which could efficiently reduce the production of false positive reports.

Keywords: SARS-CoV-2; diagnosis; digital PCR; false negative; false positive; real time PCR.

Figure 1.

Results of qRT-PCR and ddPCR for different primers/probes sets. (A) Results of qRT-PCR with different primer/probe sets. Dilution multiples (converted to log₁₀) were plotted on the X axis versus measured Ct values of qRT-PCR on the Y axis. CT value ≥40 were plotted as not detected (ND). (B) Results of ddPCR with different primer/probe sets. Dilution multiples (converted to log₁₀) were plotted on the X axis versus measured values of ddPCR (converted to log₁₀) on the Y axis. Value with 0 copies/ reaction were plotted as ND. For each primer-probe set, we show the range of measured cycle threshold and concentration values obtained with mock samples from healthy people (IgM/IgG negative) in green-shaded areas. Values of patients’ samples beyond the maximum values of mock samples were judged as positive.

Figure 1.

Results of qRT-PCR and ddPCR for different primers/probes sets. (A) Results of qRT-PCR with different primer/probe sets. Dilution multiples (converted to log₁₀) were plotted on the X axis versus measured Ct values of qRT-PCR on the Y axis. CT value ≥40 were plotted as not detected (ND). (B) Results of ddPCR with different primer/probe sets. Dilution multiples (converted to log₁₀) were plotted on the X axis versus measured values of ddPCR (converted to log₁₀) on the Y axis. Value with 0 copies/ reaction were plotted as ND. For each primer-probe set, we show the range of measured cycle threshold and concentration values obtained with mock samples from healthy people (IgM/IgG negative) in green-shaded areas. Values of patients’ samples beyond the maximum values of mock samples were judged as positive.