Background: The ongoing Coronavirus disease 2019 (COVID-19) pandemic has spread across the globe and is representing a huge challenge for all human population. Many commercial qRT-PCR assays have been developed to detect SARS-CoV-2, but related method validation especially the sensitivity evaluation has been insufficient, resulting in some false-negative cases have been reported.
Methods: The analytical sensitivity of nine brands of qRT-PCR kits for detecting SARS-CoV-2 was evaluated in parallel based on a newly developed certified reference material, which was derived from genomic RNA of SARS-CoV-2 from clinical positive specimens. After validation of the the reference material by digital PCR, the detection sensitivity of these kits was preliminarily tested using the serially diluted reference material, resulting in three kits with two significantly different sensitivity levels were selected for further evaluation. We sequenced the qRT-PCR products for assay specificity evaluation, and used serial dilutions of the reference material to calculate amplification efficiency and estimate the limit of quantification as well as 95% limit of detection..
Results: The results indicated that the analytical sensitivity varied markedly among these kits. For the three types of qRT-PCR kits (Kit-1, Kit-2 and Kit-7), specificity of the PCR products was confirmed by sequence alignment, in which the target amplicons completely matched the corresponding parts of the genome of SARS-CoV-2. The resulting limit of detection from replicate tests for the Kit-1 and Kit-2 was 5.6 copies (N), 3.5 copies (ORF 1ab), and 6.4 copies (N), 4.6 copies (ORF 1ab), respectively, at 95% probability. Compared with Kit-7, the limit of detection as well as limit of quantification of Kit-1 and Kit-2 were significantly lower, further supporting that the both kits worked well to detect low abundance of SARS-CoV-2.
Conclusions: Considering that most of the tested kits have been approved for in vitro diagnostics (IVD) in China, the established method here provides a reliable tool to evaluate the sensitivity performance of various qRT-PCR kits for SARS-CoV-2 detection and thus enhance quality control of qRT-PCR assays, improving the laboratory diagnostic capability for fighting the COVID-19 pandemic.