Development of RNA-Based Assay for Rapid Detection of SARS-CoV-2 in Clinical Samples.

INTRODUCTION: The ongoing spread of pandemic coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of growing concern. Rapid diagnosis and management of SARS-CoV-2 are crucial for controlling the outbreak in the community. Here, we report the development of a first rapid-colorimetric assay capable of detecting SARS-CoV-2 in the human nasopharyngeal RNA sample in less than 30 min. METHOD: We utilized a nanomaterial-based optical sensing platform to detect RNA-dependent RNA polymerase gene of SARS-CoV-2, where the formation of oligo probe-target hybrid led to salt-induced aggregation and change in gold-colloid color from pink to blue visibility range. Accordingly, we found a change in colloid color from pink to blue in assay containing nasopharyngeal RNA sample from the subject with clinically diagnosed COVID-19. The colloid retained pink color when the test includes samples from COVID-19 negative subjects or human papillomavirus-infected women. RESULTS: The results were validated using nasopharyngeal RNA samples from positive COVID-19 subjects (n = 136). Using real-time polymerase chain reaction as gold standard, the assay was found to have 85.29% sensitivity and 94.12% specificity. The optimized method has detection limit as little as 0.5 ng of SARS-CoV-2 RNA. CONCLUSION: We found that the developed assay rapidly detects SARS-CoV-2 RNA in clinical samples in a cost-effective manner and would be useful in pandemic management by facilitating mass screening.

Conformational flexibility and structural variability of SARS-CoV2 S protein.

Spike (S) glycoprotein of SARS-CoV2 exists chiefly in two conformations, open and closed. Most previous structural studies on S protein have been conducted at pH 8.0, but knowledge of the conformational propensities under both physiological and endosomal pH conditions is important to inform vaccine development. Our current study employed single-particle cryoelectron microscopy to visualize multiple states of open and closed conformations of S protein at physiological pH 7.4 and near-physiological pH 6.5 and pH 8.0. Propensities of open and closed conformations were found to differ with pH changes, whereby around 68% of S protein exists in open conformation at pH 7.4. Furthermore, we noticed a continuous movement in the N-terminal domain, receptor-binding domain (RBD), S2 domain, and stalk domain of S protein conformations at various pH values. Several key residues involving RBD-neutralizing epitopes are differentially exposed in each conformation. This study will assist in developing novel therapeutic measures against SARS-CoV2.