Development and validation of a PCR-free nucleic acid testing method for RNA viruses based on linear molecular beacon probes

Abstract

Abstract Background RNA viruses periodically trigger pandemics of severe human diseases, frequently causing enormous economic losses. Here, a nucleic acid extraction-free and amplification-free RNA virus testing probe was proposed for the sensitive and simple detection of classical swine fever virus (CSFV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), based on a double-stranded molecular beacon method. This RNA virus probe contains two base sequences—a recognition strand that binds to the specific domain of CSFV N2 or SARS-CoV-2 N, with a fluorophore (FAM) labeled at the 5′ end, and a complementary strand (CSFV-Probe B or SARS-CoV-2-Probe B), combined with a quencher (BHQ2) labeled at the 3′ end. Results Using linear molecular beacon probe technology, the detection limit of the RNA virus probe corresponding to CSFV and SARS-CoV-2 were as low as 0.28 nM and 0.24 nM, respectively. After CSFV E2 and SARS-CoV-2 N genes were transfected into corresponding host cells, the monitoring of RNA virus probes showed that fluorescence signals were dramatically enhanced in a concentration- and time-dependent manner. These results were supported by those of quantitative (qRT-PCR) and visualization (confocal microscopy) analyses. Furthermore, CSF-positive swine samples and simulated SARS-CoV-2 infected mouse samples were used to demonstrate their applicability for different distributions of viral nucleic acids in series tissues. Conclusions The proposed RNA virus probe could be used as a PCR-free, cost-effective, and rapid point-of-care (POC) diagnostic platform for target RNA virus detection, holding great potential for the convenient monitoring of different RNA viruses for early mass virus screening. Graphical Abstract

Keywords

• SARS-CoV-2
• CSFV
• Nucleic acid testing
• PCR-free
• Molecular beacon probes