Development and Evaluation of AccuPower? COVID-19 Multiplex Real-Time RT-PCR Kit and AccuPower? SARS-CoV-2 Multiplex Real-Time RT-PCR Kit for SARS-CoV-2 Detection in Sputum, NPS/OPS, Saliva and Pooled Samples

Abstract Rapid and accurate detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the successful control of the current global COVID-19 pandemic. The real-time reverse transcription polymerase chain reaction (Real-time RT-PCR) is the most widely used detection technique. This research describes the development of two novel multiplex real-time RT-PCR kits, AccuPower? COVID-19 Multiplex Real-Time RT-PCR Kit (NCVM) specifically designed for use with the ExiStation?48 system (comprised of ExiPrep?48 Dx and Exicycler?96 by BIONEER, Korea) for sample RNA extraction and PCR detection, and AccuPower? SARS-CoV-2 Multiplex Real-Time RT-PCR Kit (SCVM) designed to be compatible with manufacturers’ on-market PCR instruments. The limit of detection (LoD) of NCVM was 120 copies/μL and the LoD of the SCVM was 2 copies/mL for both the gene and the SARS-CoV-2 gene (N gene and RdRp gene). The AccuPower? kits demonstrated high precision with no cross reactivity to other respiratory-related microorganisms. The clinical performance of AccuPower? kits was evaluated using the following clinical samples: sputum and nasopharyngeal/oropharyngeal swab (NPS/OPS) samples. Overall agreement of the AccuPower? kits with a Food and Drug Administration (FDA) approved emergency use authorized commercial kit (STANDARD? M nCoV Real-Time Detection kit, SD BIOSENSOR, Korea) was above 95% (Cohen’s kappa coefficient ≥ 0.95), with a sensitivity of over 95%. The NPS/OPS specimen pooling experiment was conducted to verify the usability of AccuPower? kits on pooled samples and the results showed greater than 90% agreement with individual NPS/OPS samples. The clinical performance of AccuPower? kits with saliva samples was also compared with NPS/OPS samples and demonstrated over 95% agreement (Cohen’s kappa coefficient > 0.95). This study shows the BIONEER NCVM and SCVM assays are comparable with the current standard confirmation assay and are suitable for effective clinical management and control of SARS-CoV-2.