Enhanced Throughput of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Real-Time RT-PCR Panel by Assay Multiplexing and Specimen Pooling

Lu, Xiaoyan; Sakthivel, Senthilkumar K.; Wang, Lijuan; Lynch, Brian; Dollard, Sheila M.

A multiplex real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was developed based on the same primer and probe sequences of an existing U.S. CDC Emergency Use authorized test panel, targeting SARS-CoV-2 N1, N2 and human RNase P genes in singleplex. Both singleplex and multiplex assays demonstrated linear dynamic ranges of 8 orders of magnitude and analytical limits of detection of 5 RNA transcript copies/reaction. Both assays showed 100% agreement with 364 previously characterized clinical specimens (146 positive and 218 negative) for detection of SARS-CoV-2 RNA. To further increase testing throughput, 40 positive and 20 negative four-specimen pools were tested by the multiplex assay and showed 97.75% and 100% congruence with individual specimen tests, respectively. rRT-PCR assay multiplexing and sample pooling, individually or in combination, can substantially increase throughput of SARS-CoV-2 testing.