Validation of a modified CDC assay and performance comparison with the NeuMoDx™ and DiaSorin® automated assays for rapid detection of SARS-CoV-2 in respiratory specimens

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), has spread rapidly around the globe since it was first identified in December of 2019 in Wuhan, China. In a race to contain the infection, researchers and healthcare officials have developed several assays to help diagnose individuals with COVID-19. To help laboratories decide what assay to bring into testing lines, factors such as assay availability, cost, throughput, and TAT should be considered. Here we validated a modified version of the CDC assay and used it as a reference to evaluate the performance of the NeuMoDxTM SARS-CoV-2 and DiaSorin SimplexaTM Covid-19 Direct assays. In silico analysis and clinical sample testing showed that the primers/probes designed by the CDC were specific to the SARS-CoV-2 as they accurately detected all reactive samples with an assay LoD of 200 copies/mL. The performance of the three assays were analyzed using 159 nasopharyngeal swabs specimen tested within 1-5 days after routine testing. A 100 % agreement was observed between the commercial assays and the modified CDC SARS-CoV-2 assay. A deeper look at the Ct values showed no significant difference between NeuMoDx and the modified CDC SARS-CoV-2 assay, whereas DiaSorin had lower overall Ct values than the modified CDC SARS-CoV-2 assay. NeuMoDx and DiaSorin workflows were much easier to perform. NeuMoDx has the highest throughput and shortest TAT, whereas although the modified CDC SARS-CoV-2 assay has comparable throughput to DiaSorin, it has the longest hands-on time and highest TAT.

Keywords: COVID-19; Pandemic; SARS-CoV-2.

Fig. 1

A) Multiple sequence alignment of partial sequence of the nucleocapsid gene of MERS-CoV (NC 038294.1), SARS-CoV-2 (NC_045512.2), SARS-CoV (NC_004718.3) showing target regions of N1 and N2 primer/probe sets. Forward primers sequences are bolded, probes are faded and itilicized, and reverse primers are underlined and bolded. Note: 780 bases between the two primer sets were omitted to shorted the length of the sequence. B) Capilary gel electrophoresis picture generated using the Agilent DNA 7500 kit on the Agilent 2100 bioanalyzer instrument. Image showed single band for each primer set. From left to right: L (ladder:100 – 7000bp), N1, N2, and RP of aproximately 72 bp, 67 bp, and 65 bp, respectively.

Fig. 2

Real time PCR determining amplification efficiency of the primer/probe sets. Ten-fold serial dilution of 200,000 copies/μl of nCoVPC plasmid was tested. PCR linearity over 6 orders of magnitude with a limit of detection of 2 copies/μl; N1 slope of -3.05 with a correlation coefficient R2 = 0.99; N2 slope =-3.33 and R2 = 0.99.