Establishment of a Duplex Real Time PCR Method for Detection of SARS-COV-2

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.

Keywords:

• RT-PCR
• SARS-COV-2
• ORF1ab gene
• N gene

Additional information

Funding

This work was supported by the National Key R&D Program of China (SKLNBC2020-03).