Improved Molecular Diagnosis of COVID-19 by the Novel, Highly Sensitive and Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-PCR Assay Validated In Vitro and with Clinical Specimens

Abstract

39 On 31 st December 2019, the World Health Organization was informed of a cluster of cases of 40 pneumonia of unknown etiology in Wuhan, China. Subsequent investigations identified a novel 41 coronavirus, now named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 42 from the affected patients. Highly sensitive and specific laboratory diagnostics are important for 43 controlling the rapidly evolving SARS-CoV-2-associated Coronavirus Disease 2019 (COVID-44 19) epidemic. In this study, we developed and compared the performance of three novel real-time 45 RT-PCR assays targeting the RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), 46 and nucleocapsid (N) genes of SARS-CoV-2 with that of the reported RdRp-P2 assay which is 47 used in >30 European laboratories. Among the three novel assays, the COVID-19-RdRp/Hel 48 assay had the lowest limit of detection in vitro (1.8 TCID 50 /ml with genomic RNA and 11.2 RNA 49 copies/reaction with in vitro RNA transcripts). Among 273 specimens from 15 patients with 50 laboratory-confirmed COVID-19 in Hong Kong, 77 (28.2%) were positive by both the COVID-51 19-RdRp/Hel and RdRp-P2 assays. The COVID-19-RdRp/Hel assay was positive for an 52 additional 42 RdRd-P2-negative specimens [119/273 (43.6%) vs 77/273 (28.2%), P<0.001], 53including 29/120 (24.2%) respiratory tract specimens and 13/153 (8.5%) non-respiratory tract 54 specimens. The mean viral load of these specimens was 3.21×10 4 RNA copies/ml (range, 55 2.21×10 2 to 4.71×10 5 RNA copies/ml). The COVID-19-RdRp/Hel assay did not cross-react with 56 other human-pathogenic coronaviruses and respiratory pathogens in cell culture and clinical 57 specimens, whereas the RdRp-P2 assay cross-reacted with SARS-CoV in cell culture. The highly 58 sensitive and specific COVID-19-RdRp/Hel assay may help to improve the laboratory diagnosis 59 of COVID-19. 60 61 on March 16, 2020 by guest http://jcm.asm.org/ Downloaded from 4

Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study

“…We did in-house reverse transcriptase quanti tative PCR (RT-qPCR) targeting the SARS-CoV-2 RNA-dependent-RNA-polymerase-helicase gene region, as described (appendix p 1). 12 We did EIAs for SARS-CoV-2 nucleo protein (NP) and spike protein receptor binding domain (RBD), as described but with modifications. 13 Recombinant NP and spike protein RBD of SARS-CoV-2 were used for the EIAs.…”

The epidemiology, diagnosis and treatment of COVID-19

“…Three novel RT-PCR assays targeting the RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 were developed. Among the three novel assays, the COVID-19-RdRp/Hel assay had the lowest limit of detection in vitro; highly sensitive and specific assays may help to improve the laboratory diagnosis of COVID-19 [20] . The SARS-CoV E gene assay was more sensitive than the RdRp gene assay combined with the one-step RT-PCR system [21] .…”

Escalating infection control response to the rapidly evolving epidemiology of the coronavirus disease 2019 (COVID-19) due to SARS-CoV-2 in Hong Kong

“…Thermal cycling was performed at 55°C for 5 minutes for reverse transcription, followed by 95°C for 5 minutes, then 45 cycles of 95°C for 5 seconds, 60°C for 15 seconds, and 72°C for 15 seconds on the LightCycler 480 II system (Roche Diagnostics, Mannheim, Germany). The SARS-CoV-2 RNA loads in patient and environmental samples were determined using a real-time RT-PCR assay developed in house to target the SARS-CoV-2 RdRp gene 11 .…”