Abstract

To quickly and efficiently detect the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and prevent and control the spread of novel coronavirus disease (COVID-19), a highly sensitive duplex real-time PCR (RT-PCR) detection method has been established. In this study, the specificity of primers and probes were designed, respectively, according to the ORF1ab gene and N gene sequence of SARS-COV-2, and fluorescent probes were labeled with carboxyl fluorescein (FAM) and green fluorescent protein (VIC). The duplex RT-PCR method for detecting SARS-COV-2 with TaqMan probe was established, which has a limit of detection of 10 copies/µL, and the linear detection range of ORF1ab and N gene were 1.0 × 10¹-1.0 × 10⁵ copies/µL and 1.0 × 10¹-1.0 × 10⁶ copies/µL, respectively, realizing the simultaneous detection of ORF1ab and N genes in simulated SARS-COV-2 samples. The method has high sensitivity, accurate quantification, simple operation, and cost-saving, which can be used for rapid and efficient quantitative detection of SARS-COV-2.

摘要

为快速有效地检测严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 并预防和控制新型冠状病毒病 (COVID-19) 的传播，一种高灵敏度的双工实时 PCR (RT-PCR) 检测方法已经建立。本研究分别根据SARS-COV-2的ORF1ab基因和N基因序列设计引物和探针的特异性，荧光探针分别用羧基荧光素（FAM）和绿色荧光蛋白（VIC）标记。建立了TaqMan探针检测SARS-COV-2的双重RT-PCR方法，检测限为10拷贝/μL，ORF1ab和N基因的线性检测范围为1.0×10 ¹ -1.0×10 ⁵拷贝/µL 和 1.0 × 10 ¹ -1.0 × 10^分别为6拷贝/µL，实现了模拟SARS-COV-2样本中ORF1ab和N基因的同时检测。该方法灵敏度高、定量准确、操作简单、节约成本，可用于SARS-COV-2的快速、高效定量检测。