{"doi":"10.1101/2025.03.10.25323665","title":"Multiplexed Salivary miRNA Quantification for Predicting Severe COVID-19 Symptoms in Children Using Ligation-RPA Amplification Assay","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.","journal":"medRxiv","year":2025,"id":554959,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":5,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9541,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1024502,"name":"Zhikun Zhang","orcid":"0000-0002-0945-7176","position":1,"is_corresponding":false},{"id":628289,"name":"Aneesh Kshirsagar","orcid":"0000-0003-2288-5439","position":2,"is_corresponding":false},{"id":886738,"name":"Anthony J. Politza","orcid":"0009-0005-8670-2357","position":3,"is_corresponding":false},{"id":1123441,"name":"Usha Sethuraman","orcid":"0000-0002-5183-0325","position":4,"is_corresponding":false},{"id":409810,"name":"Srinivasan Suresh","orcid":"0000-0003-1717-7593","position":5,"is_corresponding":false},{"id":774155,"name":"Steven D. Hicks","orcid":"0000-0002-9579-6798","position":6,"is_corresponding":false},{"id":1452794,"name":"Feng Guo","orcid":"0000-0001-9833-9691","position":7,"is_corresponding":false},{"id":270357,"name":"Weihua Guan","orcid":"0000-0002-8435-9672","position":8,"is_corresponding":false},{"id":1024500,"name":"Md. Ahasan Ahamed","orcid":"0009-0006-9962-2994","position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-07-19T02:54:54.542303Z","pmid":"40162235","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}