摘要
目的建立并评价一种基于COYOTE■Flash20实时荧光定量PCR仪用于新型冠状病毒(SARS-CoV-2)核酸快速检测的方法。方法通过使用靶向SARS-CoV-2 ORF1ab和N基因的特异性引物探针组构建快速反应体系,并验证体系的灵敏度和特异性。同时,使用108例新型冠状病毒肺炎(COVID-19)临床样本对该方法进行应用评估。结果该检测方法无需核酸提取、手动操作时间仅用1 min,样本进、结果出,可以在30 min完成检测。该方法最低检测限为4×10^(2)拷贝/ml;与其他人类冠状病毒(包括HCoV-229E、HCoV-NL63、HCoV-OC43、HCoV-HKU1、SARS-CoV和MERS-CoV)以及其他引起呼吸系统疾病的病毒无交叉反应。临床样本应用评估显示,与常规需要核酸提取的RT-qPCR的总符合率为98.15%。结论通过对SARS-CoV-2快速荧光定量PCR方法的应用评价发现,该方法操作简便、快速、特异、灵敏,适用于多种场景即时快速检测需求。
Objective To establish and evaluate a rapid nucleic acid detection method for SARS-CoV-2 based on COYOTE®Flash20 real-time fluorescent quantitative PCR instrument.Methods A rapid reaction system was constructed by using specific primer and probe sets targeting ORF1ab and N gene of SARS-CoV-2,and the sensitivity and specificity of the system were verified.At the same time,108 clinical samples of COVID-19 were used to evaluate the application of this method.Results The detection method did not require nucleic acid extraction,and the manual operation time was only one minute.After the sample was sent to the system,the test could be completed in 30 minutes.The detection limit of this method was 4×10^(2) copies/ml.It had no cross-reactivity with other human coronaviruses(including HCoV-229E,HCoV-NL63,HCoV-OC43,HCoV-HKU1,SARS-CoV and MERS-CoV)and other respiratory viruses.The evaluation of clinical sample application showed that the total coincidence rate with the conventional RT-qPCR which required nucleic acid extraction was 98.15%.Conclusions Through the application evaluation of the rapid fluorescent quantitative PCR method of SARS-CoV-2,it was found that the method was simple,fast,specific and sensitive,and it was suitable for real-time and rapid detection needs in varieties of situations.
作者
牛培华
朱耀武
陆柔剑
彭静
朱娜
鲁艳军
王文玲
倪明
谭文杰
Niu Peihua;Zhu Yaowu;Lu Roujian;Peng Jing;Zhu Na;Lu Yanjun;Wang Wenling;Ni Ming;Tan Wenjie(Biotech Center for Viral Disease Energency,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206;Department of Inspection,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030)
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2021年第8期588-591,共4页
Chinese Journal of Microbiology and Immunology
基金
国家传染病重大专项(2018ZX10711001-001)
国防科技创新特区(19-163-12-ZD-025-001-01)。
