A Comparative Study of Real-Time RT-PCR-Based SARS-CoV-2 Detection Methods and Its Application to Human-Derived and Surface Swabbed Material

Abstract

Real-time RT-PCR remains a gold standard in the detection of various viral diseases. In the coronavirus 2019 pandemic, multiple RT-PCR-based tests were developed to screen for viral infection. As an emergency response to increasing testing demand, we established a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PCR diagnostics platform for which we compared different commercial and in-house RT-PCR protocols. Four commercial, one customized, and one in-house RT-PCR protocols were evaluated with 92 SARS-CoV-2-positive and 92 SARS-CoV-2-negative samples. Furthermore, economical and practical characteristics of these protocols were compared. In addition, a highly sensitive digital droplet PCR (ddPCR) method was developed, and application of RT-PCR and ddPCR methods on SARS-CoV-2 environmental samples was examined. Very low limits of detection (1 or 2 viral copies/μL), high sensitivities (93.6% to 97.8%), and high specificities (98.7% to 100%) for the tested RT-PCR protocols were found. Furthermore, the feasibility of downscaling two of the commercial protocols, which could optimize testing capacity, was demonstrated. Tested commercial and customized RT-PCR detection kits show very good and comparable sensitivity and specificity, and the kits could be further optimized for use on SARS-CoV-2 viral samples derived from human and surface swabbed samples.

Figure 1

Summary of different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR detection protocols. SARS-CoV-2 genome structure and coverage by different protocols are shown. Continuous line indicates relative gene coverage by the detection protocol. The Euroimmun and TF-MultiPlex, protocols were for research use only. CDC, Centers for Disease Control and Prevention; WHO, World Health Organization.

Figure 2

Limit of detection (LoD) of real-time RT-PCR and digital droplet PCR (ddPCR) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection protocols. A: LoD (viral copies per microliter) of different target genes of Centers for Disease Control and Prevention (CDC), TF-SinglePlex, TF-MultiPlex, Euroimmun, Oncobit RT-PCR, and Oncobit ddPCR SARS-CoV-2 detection protocols. B: Calculated R² values of SARS-CoV-2 detection protocols.

Figure 3

Specificity and sensitivity of real-time RT-PCR severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection protocols. A: Performance calculation (sensitivity/specificity) as well as calculation of percentage of inconclusive results of five real-time RT-PCR detection protocols [Centers for Disease Control and Prevention (CDC), TF-SinglePlex, TF-MultiPlex, Euroimmun, and Oncobit]. The Euroimmun RT-PCR detection protocol does not have the inconclusive category; inconclusive for Euroimmun equals an invalid result. B: Heatmap summarizing concordance of five real-time RT-PCR detection protocols (CDC, TF-SinglePlex, TF-MultiPlex, Euroimmun, and Oncobit) for both sensitivity (bottom) and specificity (top) sample cohorts.

Figure 4

Downscaling of the Centers for Disease Control and Prevention (CDC) and TF-MultiPlex protocols. A: Heatmap summarizing results of standard and downscaled protocol (CDC and TF-MultiPlex). For the CDC protocol, a RT-PCR result was defined inconclusive if RT-PCR was positive for only N1 (±N3) or for only N2 (±N3). For TF-MultiPlex a RT-PCR result was considered inconclusive if only one of the viral genes was positive. B: Limit of detection (LoD) (copies per microliter) and R² values of downscaled protocols (CDC and TF-MultiPlex). NTC, nontemplate control; PC, positive control.