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

Acute 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. RCNPM 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. RCNPM 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. RCNPM 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.