Department of Microbiology, Queen Mary Hospital, HKSAR, Hong Kong, China.
Genomics and Bioinformatics Programme, The Chinese University of Hong Kong, HKSAR, Hong Kong, China.
Int J Mol Sci. 2020 Apr 8;21(7):2574. doi: 10.3390/ijms21072574.
The pandemic novel coronavirus infection, Coronavirus Disease 2019 (COVID-19), has affected at least 190 countries or territories, with 465,915 confirmed cases and 21,031 deaths. In a containment-based strategy, rapid, sensitive and specific testing is important in epidemiological control and clinical management. Using 96 SARS-CoV-2 and 104 non-SARS-CoV-2 coronavirus genomes and our in-house program, GolayMetaMiner, four specific regions longer than 50 nucleotides in the SARS-CoV-2 genome were identified. Primers were designed to target the longest and previously untargeted nsp2 region and optimized as a probe-free real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. The new COVID-19-nsp2 assay had a limit of detection (LOD) of 1.8 TCID/mL and did not amplify other human-pathogenic coronaviruses and respiratory viruses. Assay reproducibility in terms of cycle threshold (Cp) values was satisfactory, with the total imprecision (% CV) values well below 5%. Evaluation of the new assay using 59 clinical specimens from 14 confirmed cases showed 100% concordance with our previously developed COVID-19-RdRp/Hel reference assay. A rapid, sensitive, SARS-CoV-2-specific real-time RT-PCR assay, COVID-19-nsp2, was developed.
新型冠状病毒肺炎（COVID-19）疫情已影响至少 190 个国家或地区，确诊病例 465915 例，死亡 21031 例。在以遏制为主的策略中，快速、敏感和特异的检测对于流行病学控制和临床管理非常重要。利用 96 株 SARS-CoV-2 和 104 株非 SARS-CoV-2 冠状病毒基因组，以及我们的内部程序 GolayMetaMiner，鉴定出了 SARS-CoV-2 基因组中 4 个长度超过 50 个核苷酸的特定区域。设计引物靶向最长和以前未靶向的 nsp2 区域，并将其优化为无探针实时逆转录-聚合酶链反应（RT-PCR）检测。新型 COVID-19-nsp2 检测法的检测限（LOD）为 1.8 TCID/mL，不扩增其他人类致病性冠状病毒和呼吸道病毒。就循环阈值（Cp）值而言，检测法的重现性令人满意，总不精密度（%CV）值远低于 5%。使用来自 14 例确诊病例的 59 份临床标本对新检测法进行评估，与我们之前开发的 COVID-19-RdRp/Hel 参考检测法的一致性为 100%。开发了一种快速、敏感、针对 SARS-CoV-2 的实时 RT-PCR 检测法 COVID-19-nsp2。
