Comparison of seven commercial RT-PCR diagnostic kits for COVID-19

Abstract

The final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed. The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene). We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95 % limit of detection (LOD95). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n = 13) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n = 6) and a panel of RNA from related human coronaviruses to evaluate assay specificity. PCR efficiency was ≥96 % for all assays and the estimated LOD95 varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene. We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.

Keywords: COVID-19; Coronavirus; In vitro diagnostics; RT-PCR; SARS-CoV-2; nCoV-2019.

Fig. 1

PCR efficiency for seven commercially available RT-PCR kits for the detection of SARS-CoV-2 RNA. PCR efficiency (E) for each target gene was assessed using a duplicate 10-fold dilution series of SARS-CoV-2 viral RNA. Linear regression was performed in Graphpad Prism to obtain the slope and R². The percentage efficiency was calculated from the slope using the formula E = 100*(-1 + 10^−1/slope). E-gene, gene encoding the envelope protein of SARS-CoV-2; RdRp, RNA-dependent RNA polymerase of SARS-CoV-2; N, nucleocapsid protein of SARS-CoV-2; ORF1ab, open reading frame 1a and b of SARS-CoV-2, includes the RdRp; RNA-dependent RNA polymerase of SARS-CoV-2, part of ORF1ab; RT-PCR, reverse-transcriptase polymerase chain reaction; S, spike protein of SARS-CoV-2; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Fig. 2

Different RT-PCR kits showed variations in detection rate and Ct values. RNA isolated from stored SARS-CoV-2-positive clinical samples using the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche) was subjected to the various RT-PCR assays according to the manufacturer’s instructions for use, on a LightCycler 480 II (Roche). A) Graph depicts Ct values obtained for all clinical samples (n = 13) in all RT-PCR assays. Data points above the horizontal dotted line are negative, for plotting purposes indicated with Ct 42.5. The detection rate of the complete RT-PCR kit is indicated below the data points, e.g. 10/13 means 10 out of 13 samples tested positive according to the instructions for data interpretation provided by the manufacturer. For the Seegene kit, one sample was “inconclusive” according to the manufacturer’s guide for interpretation and was therefore counted as “negative”, although a signal was observed for at least one target. B) Graph depicts only data for those clinical samples (n = 10) with the highest concentration of SARS-CoV-2 RNA and which were positively identified by all RT-PCR assays. The lines show the mean Ct value for each assay, triangles show the Ct values of the samples with the highest (sample 1) and lowest (sample 10) concentration according to the in-house E-gene PCR. E, envelope protein of SARS-CoV-2; RdRp, RNA-dependent RNA polymerase of SARS-CoV-2; N, nucleocapsid protein of SARS-CoV-2; ORF1ab, open reading frame 1a and b of SARS-CoV-2, includes the RdRp; RNA-dependent RNA polymerase of SARS-CoV-2, part of ORF1ab; RT-PCR, reverse-transcriptase polymerase chain reaction; S, spike protein of SARS-CoV-2; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.