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
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
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DOI：
10.1128/jcm.00310-20
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发表时间：
2020-05-01
影响因子：
9.4
通讯作者：
Yuen, Kwok-Yung
中科院分区：
文献类型：
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作者：
Chan, Jasper Fuk-Woo;Yip, Cyril Chik-Yan;Yuen, Kwok-Yung
2019年12月31日，世界卫生组织获悉中国武汉出现不明原因肺炎聚集性病例。随后的调查从受影响的患者身上发现了一种新型冠状病毒，现命名为严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2)。高度敏感和特异性的实验室诊断对于控制快速演变的 SARS-CoV-2 相关冠状病毒病 2019 (COVID-19) 流行非常重要。在本研究中，我们开发了三种针对 SARS-CoV-2 的 RNA 依赖性 RNA 聚合酶 (RdRp)/解旋酶 (Hel)、刺突 (S) 和核衣壳 (N) 基因的新型实时逆转录 PCR (RT-PCR) 检测方法，并将其性能与已报道的 RdRp-P2 检测方法的性能进行了比较，该方法在超过 30 个欧洲实验室中使用。在这三种新型检测中，COVID-19-RdRp/Hel 检测的体外检测限最低（基因组 RNA 为 1.8 50% 组织培养感染剂量 [TCID50]/ml，体外 RNA 转录物为 11.2 RNA 拷贝/反应）。在香港 15 名实验室确诊的 COVID-19 患者的 273 份样本中，77 份 (28.2%) 的 COVID-19-RdRp/Hel 和 RdRp-P2 检测结果均呈阳性。另外 42 份 RdRp-P2 阴性标本的 COVID-19RdRp/Hel 检测结果呈阳性（119/ 273 [43.6%] 对比 77/273 [28.2%]；P < 0.001），其中包括 29/120 (24.2%) 呼吸道标本和 13/153 (8.5%) 非呼吸道标本。这些样本的平均病毒载量为 3.21 x 10(4) RNA 拷贝/ml（范围为 2.21 x 10(2) 至 4.71 x 10(5) RNA 拷贝/ml）。 COVID-19-RdRp/Hel 检测不与细胞培养物和临床标本中的其他人类致病性冠状病毒和呼吸道病原体发生交叉反应，而 RdRp-P2 检测则与细胞培养物中的 SARS-CoV 发生交叉反应。高度敏感和特异性的 COVID-19-RdRp/Hel 检测可能有助于改善 COVID-19 的实验室诊断。
On 31 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 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 reverse transcription-PCR (RT-PCR) assays targeting the RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 with that of the reported RdRp-P2 assay, which is used in >= 30 European laboratories. Among the three novel assays, the COVID-19-RdRp/Hel assay had the lowest limit of detection in vitro (1.8 50% tissue culture infective doses [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-RdRp/Hel and RdRp-P2 assays. The COVID-19RdRp/Hel assay was positive for an additional 42 RdRp-P2-negative specimens (119/ 273 [43.6%] versus 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 3.21 x 10(4) RNA copies/ml (range, 2.21 x 10(2) to 4.71 x 10(5) RNA copies/ml). The COVID-19-RdRp/Hel assay did not cross-react with other human-pathogenic coronaviruses and respiratory pathogens in cell culture and clinical specimens, whereas the RdRp-P2 assay cross-reacted with SARS-CoV in cell culture. The highly sensitive and specific COVID-19-RdRp/Hel assay may help to improve the laboratory diagnosis of COVID-19.