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/mL and the LoD of the SCVM was 2 copies/μL for both the Pan-sarbecovirus gene and the SARS-CoV-2 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.

Improving the detection capability and efficiency of SARS-CoV-2 RNA specimens by the specimen turn-around process with multi-department cooperation

“…Nowadays, the detection of virus RNA by RT-PCR is the most widely used detection technique for confirming SARS-CoV-2 infection. As previously reported in many literature, NPS and OPS were the most widely used upper respiratory tract specimens recommended for diagnosing SARS-CoV-2 RNA with RT-PCR (21)(22)(23). There was controversy in the literature about the ability of the two sampling methods to detect viruses (24)(25)(26)(27).…”

Extraction Bottleneck in the Diagnosis of SARS-CoV-2: Evaluation of an Alternative Protocol Derived from Veterinary Use

“…The COVID-19 pandemic became the catalyst for the development of accurate detection methods for SARS-CoV-2 in order to better support the clinicians and front-line healthcare professionals. However, development of effective vaccines and the availability of high-quality diagnostic methods remain essential [ 17 , 18 ]. Currently, one of the critical points remains the bottleneck of nucleic acid extraction kits, also due to the spread of the different and more transmissible newest SARS-CoV-2 lineages (BA.4, BA.5, BQ.1, XXB).…”

Validation of SARS-CoV-2 pooled testing for surveillance using the Panther Fusion® system: Impact of pool size, automation, and assay chemistry

“…Published LLODs from other studies range from 83–288 cp/mL [ 26 , 27 ] for Aptima ® and from 62.5–125 cp/mL [ 28 , 29 ] for Fusion ® . Our analysis of LLOD for the Hologic SARS-CoV-2 assays (55–228 cp/mL for Aptima ® and 108–130 cp/mL for Fusion ® , Fig 5 ) agree with published ranges.…”