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Updated: Sep 16, 2025
Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses
Published on: November 10, 2023
Multiplexed Salivary miRNA Quantification for Predicting Severe COVID-19 Symptoms in Children Using Ligation-RPA
Md Ahasan Ahamed1,2, Zhikun Zhang2, Aneesh Kshirsagar2
1Department of Intelligent Systems Engineering, Luddy School of Informatics, Computing, and Engineering, Indiana University, Bloomington, Indiana 47408, United States.
Insights
A new portable assay accurately detects salivary microRNA (miRNA) levels in children with COVID-19. This rapid, noninvasive method identifies severe cases by detecting specific miRNA downregulation, aiding early intervention.
Area of Science:
- Biomarkers
- Molecular Diagnostics
- Point-of-Care Testing
Background:
- COVID-19 can cause severe complications in children, necessitating early risk identification.
- Salivary microRNA (miRNA) alterations are potential biomarkers for predicting severe COVID-19 outcomes.
- Existing methods for miRNA detection lack speed, portability, or quantitative accuracy.
Purpose of the Study:
- To develop a rapid, noninvasive, and quantitative assay for detecting salivary miRNA expression in children with COVID-19.
- To establish a portable platform for multiplex miRNA detection as an alternative to sequencing.
- To assess the diagnostic accuracy of the developed assay for identifying severe COVID-19 cases.
Main Methods:
- Developed a ligation-coupled recombinase polymerase amplification (RPA) assay for multiplex miRNA detection.
- Utilized miR-1273, miR-296, and miR-29 as target miRNAs.
- Validated portable RNA extraction against benchtop methods and tested the assay on 154 clinical samples.
Main Results:
- The assay demonstrated high sensitivity (1 fM detection limit) and 100% specificity.
- Portable extraction methods showed strong correlation with benchtop methods (R² > 0.85, r > 0.92).
- Severe COVID-19 cases exhibited significant miRNA downregulation compared to nonsevere cases, with an AUC of 0.98.
Conclusions:
- The developed ligation-coupled RPA assay is a highly accurate and sensitive tool for detecting salivary miRNA biomarkers.
- This portable platform enables rapid, noninvasive diagnosis of severe COVID-19 in children, especially in point-of-care settings.
- The assay facilitates informed clinical decisions, resource optimization, and improved patient outcomes.
Abstract:
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 R2 > 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.
