ArticleJournal of translational medicine2026
Development of a one-tube PAM-independent RCNPM platform using Cas12a for ultra-rapid simultaneous miR-499 and cTnT detection in early acute myocardial infarction diagnosis.
Article in Journal of translational medicine, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
backgroundAcute myocardial infarction (AMI) benefits from biomarkers and assays that support early triage. Cardiac troponin T (cTnT) is widely used, but may be less informative early after ischemia and can be elevated in non-AMI conditions. Circulating miR-499 has been reported to rise early after myocardial injury, yet miRNA detection is complicated by short target length and PAM constraints in many CRISPR-Cas12a designs. We developed a PAM-independent, one-tube isothermal RT–RPA–Cas12a platform (RCNPM) for miR-499 and evaluated its analytical performance and clinical discrimination, with routine clinical cTnT analyzed in parallel.
methodsRCNPM integrates stem-loop reverse transcription, recombinase polymerase amplification (RPA), and Cas12a trans-cleavage fluorescence readout at 37 °C in a single vessel. Primers and reaction components were screened/optimized, and specificity was assessed using non-target miRNAs. Clinical evaluation was performed in a single-center prospective cohort (n = 150; 75 AMI meeting ESC criteria, 75 controls) with random coding and blinded miR-499 testing. RCNPM was benchmarked against RT–qPCR (Spearman correlation; Bland–Altman). Serum cTnT was obtained from routine hospital immunoassay testing and was not measured within the RCNPM reaction. ROC analyses were performed with 95% CIs.
resultsRCNPM detected miR-499 down to 1 aM and showed no detectable signal in the non-target miRNA panel. The RT–RPA–Cas12a step required ~ 30 min (workflow including extraction ~ 45–60 min). In the cohort, RCNPM agreed with RT–qPCR (Spearman r = 0.96, p < 0.001) with minimal mean bias. In this dataset, ROC analyses yielded AUC = 1.00 (95% CI 1.00–1.00) for miR-499 (RCNPM and RT–qPCR) and routine cTnT.
conclusionsRCNPM enables PAM-independent, one-tube isothermal miR-499 detection with strong agreement to RT–qPCR in a single-center cohort. External validation in larger, multi-center and more heterogeneous disease-control cohorts is warranted.
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