On 31(st) December 2019, the World Health Organization was informed of a cluster of cases of pneumonia of unknown etiology in Wuhan, China. Subsequent investigations identified a novel coronavirus, now named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from the affected patients. Highly sensitive and specific laboratory diagnostics are important for controlling the rapidly evolving SARS-CoV-2-associated Coronavirus Disease 2019 (COVID-19) epidemic. In this study, we developed and compared the performance of three novel real-time RT-PCR assays targeting the RNA-dependent RNA polymerase (Rd. Rp)/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 with that of the reported Rd. Rp-P2 assay which is used in >30 European laboratories. Among the three novel assays, the COVID-19-Rd. Rp/Hel assay had the lowest limit of detection in vitro (1.8 TCID50/ml with genomic RNA and 11.2 RNA copies/reaction with in vitro RNA transcripts). Among 273 specimens from 15 patients with laboratory-confirmed COVID-19 in Hong Kong, 77 (28.2%) were positive by both the COVID-19-Rd. Rp/Hel and Rd. Rp-P2 assays. The COVID-19-Rd. Rp/Hel assay was positive for an additional 42 Rd. Rd-P2-negative specimens [119/273 (43.6%) vs 77/273 (28.2%), P<0.001], including 29/120 (24.2%) respiratory tract specimens and 13/153 (8.5%) non-respiratory tract specimens. The mean viral load of these specimens was 3.21x10(4) RNA copies/ml (range, 2.21x10(2) to 4.71x10(5) RNA copies/ml). The COVID-19-Rd. Rp/Hel assay did not cross-react with other human-pathogenic coronaviruses and respiratory pathogens in cell culture and clinical specimens, whereas the Rd. Rp-P2 assay cross-reacted with SARS-CoV in cell culture. The highly sensitive and specific COVID-19-Rd. Rp/Hel assay may help to improve the laboratory diagnosis of COVID-19.
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