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bioRxiv · 10.1101/2020.06.04.135608

Design of a Novel Multiplex Real Time RT-PCR Assay for SARS-CoV-2 Detection

Zhen, W.·Berry, G. J.
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Abstract

Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 386,000 deaths globally as of June 4, 2020. In this study, we developed a novel multiplex real time reverse transcription (RT)-PCR test for detection of SARS-CoV-2, with primers designed to amplify a 108 bp target on the spike surface glycoprotein (S gene) of SARS-CoV-2 and a hydrolysis Taqman probe designed to specifically detect SARS-CoV-2. Following our design, we evaluated the Limit of detection (LOD) and clinical performance of this laboratory-developed test (LDT). A LOD study with inactivated whole virus exhibited equal performance to that seen in the modified CDC assay with a final LOD of 1,301 ± 13 genome equivalents/ml for our assay vs 1,249 ± 14 genome equivalents/ml for the modified CDC assay. In addition, a clinical evaluation with 270 nasopharyngeal (NP) swab specimens exhibited 98.5% positive percent agreement and 99.3% negative percent agreement with the modified CDC assay. The multiplex design of this assay allows the 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 have been critical. Our results demonstrate that our multiplex assay performs as well as the modified CDC assay, but is more efficient and cost effective and is therefore adequate for use as a diagnostic assay and for epidemiological surveillance and clinical management of SARS-CoV-2.View Full Text

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Zhen, W., Berry, G. J.. 2020-06-05. Design of a Novel Multiplex Real Time RT-PCR Assay for SARS-CoV-2 Detection. https://doi.org/10.1101/2020.06.04.135608

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