Comparison of eleven in vitro diagnostic assays for the detection of SARS-CoV-2 RNA

Abstract

Transmission mitigation of SARS-CoV-2 requires the availability of accurate and sensitive detection methods. There are several commercial ad hoc molecular diagnostic kits currently on the market, many of which have been evaluated by different groups. However, in low resource settings the availability and cost of these commercial kits can be a limiting factor for many diagnostic laboratories. In such cases alternatives need to be identified. With this in mind, eight commercial reverse transcription quantitative real-time PCR (RT-qPCR) master mixes from Applied Biosystems (Thermo Fisher Scientific), Bio-Rad, Biotech Rabbit, Promega, Qiagen, QuantaBio, Invitrogen (Thermo Fisher Scientific) and Takara using the same commercial primer and probe mix [LightMix® Modular SARS and Wuhan CoV E-gene mix (TIB MolBiol, Germany)] were evaluated. Three ad hoc molecular diagnostic kits [GeneFinder™ COVID-19 Plus RealAmp kit (Osang Healthcare); genesig® Real-Time PCR Coronavirus COVID-19 (Primerdesign); and ViroReal® Kit SARS-CoV-2 & SARS-CoV (Ingenetix)] were also included in the study. The limit of detection was calculated for each assay using serial dilutions of a defined clinical sample. The performances of the assays were compared using a panel of 178 clinical samples and their analytical specificity assessed against a panel of human betacoronaviruses. Inter assay agreement was assessed using statistical tests (Bland-Altman, Fleiss-Kappa and Cohen's Kappa) and was shown to be excellent to good in all cases. We conclude that all of the assays evaluated in this study can be used for the routine detection of SARS-CoV-2 and that the RT-qPCR master mixes are a valid alternative to ad hoc molecular diagnostic kits.

Keywords: Agreement; Evaluation; Limit of detection; Master mixes; Molecular diagnostic assays; SARS-CoV-2.

Fig. 1

Distribution and variability in the Cq values of samples across various test assays. Nucleic acid extracts from 178 clinical samples were tested using eleven assays. The negative amplifications were arbitrarily given the Cq values of 45 for plotting purposes allowing for their visualization above the blue line (set to Cq = 43) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

Fig. 2

Bland-Altman plots comparing the WRP to three representative assays. The plots show differences between the Cq values of the WRP and the tested assays against the average of the Cq values: A.WRP vs One-Step RT-PCR (Qiagen), showing an example of almost perfect agreement; B.WRP vs. iTaq™ Universal Probes One-Step Kit (Bio-Rad), showing an example of a tested assay with lower Cq values than the WRP; C.WRP vs GeneFinder™ COVID-19 Plus RealAmp kit (Primerdesign Ltd) showing an example of a tested assay with higher Cq values than the WRP. The red dotted lines represent the lines of identity (i.e. perfect agreement). The grey lines represent the bias between the test assays and the WRP. The grey dotted lines represent the limits (upper and lower) of agreements. Bland-Altman plots for all the tested assays are illustrated in the supplemental material (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).