Clinical evaluation of a multiplex RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples

Abstract

The aim of this study was to identify and validate a sensitive, high-throughput and cost-effective SARS-CoV-2 RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a University surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The EZ-SARS-CoV-2 RT-PCR assay analytical sensitivity was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. Clinical performance of the EZ-SARS-CoV-2 assay was determined using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94% and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by Cohen’s kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity, excellent performance in AN sample matrix and in pooled upper respiratory samples support its use in a high-throughput surveillance testing program.