Rapid detection of plasma exosomal LncRNA CASC9 for HCC using RT-RPA-CRISPR/Cas12a assay.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. Early detection is essential for improving patient outcomes. Long non-coding RNAs (lncRNAs) in plasma exosomes have emerged as promising non-invasive biomarkers. However, sensitive detection methods remain limited. Plasma exosomes were isolated and validated using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot (WB). RNA sequencing identified CASC9 as the most significantly upregulated exosomal lncRNA in HCC patients. Its diagnostic value was evaluated using real-time quantitative PCR (RT-qPCR) and a novel RT-RPA-CRISPR/Cas12a fluorescence assay. Diagnostic performance was assessed through receiver operating characteristic (ROC) curve analysis and compared with alpha-fetoprotein (AFP). Exosomal CASC9 levels were significantly elevated in HCC patients and correlated with tumor size, stage, and number (P < 0.001). ROC analysis demonstrated that CASC9 had superior diagnostic accuracy (area under the curve [AUC] = 0.822) compared to AFP (AUC = 0.795), with further improvement when combined (AUC = 0.875). The RT-RPA-CRISPR/Cas12a assay achieved a detection limit of 0.1 copies/μL, outperforming RT-qPCR. When combined with RT-qPCR and AFP, the method achieved an AUC of 0.987 against normal controls and 0.975 against benign cases. Plasma exosomal CASC9 is a promising diagnostic biomarker for HCC. The RT-RPA-CRISPR/Cas12a assay offers a rapid, ultra-sensitive, and clinically feasible detection strategy.