一种基于荧光qPCR检测新型冠状病毒核酸的优化反应体系的建立及相关测试

Optimization of a fluorescent qPCR detection for RNA of SARS-CoV-2

摘要:

为实时并快速地检出SARS-CoV-2冠状病毒RNA，对基于荧光定量聚合酶链式反应 (Polymerase chain reaction，PCR) 的反应体系进行了优化。结果表明，按照本实验提供的方法操作后，检测SARS-CoV-2冠状病毒所用的RNA样本的最小浓度稀释度可调至1/10 000 (初始值设为10 ng/μL)。且用于检测COVID-19临床阳性样本所测得循环值 (Cycle threshold，Ct) 均低于35或40。其灵敏性测试结果也表明该方法的敏感性较好。同时在同等条件下，与目前市场上的COVID-19试剂盒的检测结果基本一致，并且检测循环数缩短2个单位。因此，本实验所建立的体系适用于前期临床诊断的筛查工作，为在医学上实现快速诊断提供了工具。

关键词:新型冠状病毒感染的肺炎-2019 (COVID-19)，严重急性呼吸系统综合症冠状病毒2 (SARS-CoV-2)，N基因，ORF1a 基因，荧光实时定量PCR

Abstract:

We optimized a fluorescent quantitative polymerase chain reaction (qPCR) assay system for rapid and real time detection of SARS-CoV-2 RNA. The results show that the lowest dilution of RNA samples used for the detection of SARS-CoV-2 RNA could reach 1/10 000 (the initial value is set as 10 ng/μL). Moreover, the cycle threshold (Ct) for samples of clinically diagnosed COVID-19 was lower than 35 or 40. The sensitivity of this method was satisfactory. The results were consistent with those of the COVID-19 detection kit on the market under the same conditions, but the number of cycles required was shortened by about 2. Therefore, the optimized assay developed in this study can be used in screening and early clinical diagnosis. Our work provides a tool to facilitate rapid clinical diagnosis of COVID-19.

Key words:COVID-19, SARS-CoV-2, N gene, ORF1a gene, fluorescence real-time quantitative PCR