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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
Insights
A new portable assay rapidly detects salivary microRNA (miRNA) biomarkers for predicting severe COVID-19 in children. This non-invasive method aids early risk identification and clinical decision-making, especially in resource-limited settings.
Area of Science:
- Biochemistry
- Molecular Biology
- Pediatric Infectious Diseases
Background:
- SARS-CoV-2 can cause severe complications in children, necessitating early identification of high-risk cases.
- Salivary microRNA (miRNA) alterations are potential biomarkers for predicting COVID-19 severity.
- Current miRNA quantification methods like sequencing are not rapid or non-invasive, limiting clinical utility.
Purpose of the Study:
- To develop a rapid, specific, and sensitive non-invasive assay for quantifying salivary miRNA biomarkers to predict severe COVID-19 in children.
- To establish a portable platform for miRNA quantification as an alternative to traditional sequencing methods.
Main Methods:
- Development of a ligation-recombinase polymerase amplification (RPA) assay targeting specific salivary miRNAs (miR-1273, miR-296, miR-29).
- Validation of portable RNA extraction against benchtop methods using clinical samples (R-square > 0.85, r > 0.92).
- Quantification of miRNA levels in 154 clinical samples to assess diagnostic accuracy (AUC of 0.98).
Main Results:
- The ligation-RPA assay demonstrated high sensitivity (quantifying down to 1 fM) and 100% specificity.
- Portable extraction methods showed strong correlation with benchtop methods.
- Significant downregulation of specific miRNAs was observed in severe COVID-19 cases compared to non-severe cases.
Conclusions:
- The developed portable ligation-RPA assay is a highly accurate and sensitive tool for non-invasively predicting severe COVID-19 in children using salivary miRNA biomarkers.
- This platform facilitates timely clinical decisions and resource optimization, particularly in resource-limited settings.
Abstract:
Most children with SARS-CoV-2 have mild or asymptomatic symptoms, but some develop severe complications. Early identification of high-risk cases is crucial for timely intervention. Alterations in salivary microRNA (miRNA) levels serve as biomarkers for severity prediction. However, a rapid, non-invasive method is needed to quantify miRNA level changes as an alternative to sequencing. Here, we developed a highly specific and sensitive ligation-recombinase polymerase amplification (RPA) assay for quantifying severe and non-severe miRNAs on a portable platform. The assay begins with a miRNA-templated annealing and ligation-RPA reaction of miR-1273, miR-296, and miR-29. We quantified 100 pM to 1 fM, resolving 1 fM, with 100% specificity. Next, we validated portable extraction against benchtop extraction, achieving R-square > 0.85 and r > 0.92 in clinical samples. Finally, testing 154 clinical samples revealed severe miRNA downregulation compared to non-severe cases. The assay achieved high diagnostic accuracy with an AUC of 0.98. This platform enables informed clinical decisions and optimizes resources, especially in resource-limited settings.
