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

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.

Automated summary

A nucleic acid extraction-free and amplification-free RNA virus testing probe based on the double-stranded molecular beacon method is proposed for the sensitive and simple detection of CSFV and SARS-CoV-2. The detection limit of the probe was found to be as low as 0.28 nM and 0.24 nM for CSFV and SARS-CoV-2, respectively. The probe showed enhanced fluorescence signals in a concentration- and time-dependent manner when CSFV E2 and SARS-CoV-2 N genes were transfected into host cells. The results were supported by quantitative and visualization analyses. The proposed RNA virus probe could serve as a PCR-free, cost-effective, and rapid diagnostic platform for target RNA virus detection.