Abstract
This research describes the development of a new multiplex real-time RT-PCR test for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with primers designed to amplify a 108 bp target on the spike surface glycoprotein (S gene) and a hydrolysis TaqMan probe designed to specifically detect SARS-CoV-2. The limit of detection (LOD) and clinical performance of this new assay were evaluated. A LOD study with inactivated virus exhibited performance equal to the modified CDC assay, with a final LOD of 1301 ± 13 genome equivalents/mL for the Northwell Health Laboratories laboratory-developed test (NWHL LDT) versus 1249 ± 14 genome equivalents/mL for the modified CDC assay. In addition, a clinical evaluation with 270 nasopharyngeal swab specimens exhibited 98.5% positive percent agreement and 99.3% negative percent agreement compared with the modified CDC assay. The NWHL LDT multiplex design allows testing of 91 patients per plate, versus a maximum of 29 patients per plate on the modified CDC assay, providing the benefit of testing significantly more patients per run and saving reagents, during a time when both of these parameters are critical. The results show that the NWHL LDT multiplex assay performs as well as the modified CDC assay but is more efficient and cost-effective and can be used as a diagnostic assay and for epidemiologic surveillance and clinical management of SARS-CoV-2.