Multiplexed Salivary miRNA Quantification for Predicting Severe COVID-19 Symptoms in Children Using Ligation-RPA Amplification Assay

While most children with COVID-19 experience mild symptoms or remain asymptomatic, some may develop severe complications. Early identification of children at risk for severe outcomes is essential to ensuring timely and effective intervention. Recent studies have identified alterations in salivary microRNA (miRNA) expression levels as promising biomarkers for predicting severe complications in children. However, there remains a need for a rapid, noninvasive, and quantitative method to detect miRNA expression level changes, as their upregulation or downregulation serves as a hallmark of various diseases, providing an alternative to sequencing-based methods. Here, we developed a highly specific and sensitive ligation-coupled recombinase polymerase amplification (RPA) assay for quantitatively detecting multiplex severe and nonsevere miRNAs on a portable platform. The assay begins with an miRNA-templated annealing and ligation reaction of miR-1273, miR-296, and miR-29, followed by an RPA reaction. We quantified 100 pM to 1 fM, resolving 1 fM, with 100% specificity. Next, we validated portable extraction against benchtop extraction, achieving R 2 > 0.85 and r > 0.92 in clinical samples. Finally, testing 154 clinical samples revealed severe miRNA downregulation compared to nonsevere cases. The assay achieved high diagnostic accuracy with an area under the curve (AUC) of 0.98. This platform would empower clinicians to make informed decisions, optimize resource allocation, and improve outcomes, particularly in point-of-care (POC) settings.