SARS-CoV-2 genome RNA detection based on multiplex Real-Time RT-PCR assay

Objectives: This work aimed to develop a multiplex Real-Time RT-PCR assay to detect the SARS-CoV-2 virus in a biological sample. Methods: Multiplex Real-Time one-step RT-PCR was performed compared with the singleplex CDC protocol. For the detection of the N1 and N2 viral regions and host human RNase P gene, the fluorescent dyes VIC-BHQ-1, FAM-BHQ-1, and TAMRA-BHQ-2, respectively, were tested in a one-tube triplex reaction. The swabs from 334 nasopharyngeal samples were tested in comparisons with the singleplex method, and a pUC18 plasmid was constructed with concatenating regions from nucleocapsid and RNAse P gene as a positive control. Results: Paired data between the two assays presented a positive correlation. Comparative data demonstrated that the qRT-PCR multiplex was an efficient method presenting 83% of concordance with the singleplex, and the RNA from SARS-CoV-2 was detected in 31 and 41%, respectively. In addition, in 3% of the samples, the viral RNA was only detected in the multiplex. Conclusions: A fast, accurate, and low-cost method for detecting SARS-CoV-2 was obtained, highlighting the choice of conventional fluorophores and probes assisting in epidemiological surveillance and the clinical management of this serious public health disease.