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Article Abstract
This study presents a novel method for direct amplification of multiple microRNAs (miRNAs) from serum samples using Sensitive and Multiplexed One-Step RT-qPCR (SMOS-qPCR). The technique eliminates the need for separate miRNA extraction and purification steps, offering a streamlined approach for non-invasive early disease diagnosis. We optimized reaction conditions, including serum treatment methods and PCR system volumes, to enhance interference resistance and detection sensitivity. The optimized serum direct SMOS-qPCR demonstrated a detection limit as low as 6 × 10 copies per μL for single-target miRNA, with excellent amplification efficiency ( > 0.99). In multiplex detection, the method successfully quantified four miRNAs simultaneously, maintaining high sensitivity and reproducibility. Analysis of 20 clinical serum samples further validated the method's applicability for large-scale screening. Overall, this rapid, cost-effective, and user-friendly approach represents a significant advancement in miRNA detection technology, potentially facilitating earlier and more accessible disease diagnosis.
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