Abstract

The development of rapid, highly sensitive, and specific nucleic acid detection methods for pathogens is crucial for effective control of communicable diseases. Current techniques, however, are often limited by the high costs associated with synthesizing multiple target-specific fluorescent probes and the reliance on expensive nucleases. To address these limitations, we herein propose a double-stranded universal probe (ds-Uprobe)-mediated nucleic acid detection strategy. By strategically modifying the forward primer with a universal tag sequence and designing a corresponding ds-Uprobe, we successfully established ds-Uprobe-mediated PCR and LAMP assays. This marks a significant advancement where a single probe is universally applicable for detecting diverse nucleic acid targets. The universal ds-Uprobe strategy showed a wide detection range in PCR (from 101/102 to 106 copies/μL) and LAMP (from 102 to 106 copies/μL), with a limit of detection ranging from 10 copies/μL to 100 copies/μL. Furthermore, results from clinical samples showed high consistency and concordance with the gold standard qPCR, confirming the clinical viability and reliability of the ds-Uprobe-mediated assays. This simple, cost-effective, and highly versatile strategy enables sensitive and accurate detection and holds substantial promise for broader integration into various nucleic acid amplification tests for molecular diagnostics.