DBS Direct Elution and Multiplex Digital PCR for SMN1 and SMN2 Copy-Number and CFTR Variant Detection: Development and Validation of a Molecular Screening Workflow

Background/Objectives: Scalable molecular workflows may facilitate carrier screening for spinal muscular atrophy (SMA) and cystic fibrosis (CF). This study aimed to develop and validate a dried blood spot (DBS)-based multiplex digital PCR (dPCR) workflow for SMN1 and SMN2 copy-number analysis and targeted detection of CFTR p.Phe508del. Methods: The workflow combined direct DNA elution with multiplex dPCR. Analytical validation used 50 previously characterized samples, with multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing as reference methods. The workflow was subsequently applied to 1670 women from Crete and Ikaria, Greece, followed by targeted testing of 10 partners. Results: All 50 directly eluted samples amplified successfully. dPCR showed complete agreement with reference methods. Repeatability coefficients of variation were below 5%. Copy-number classification performance estimates are apparent, as thresholds were derived and evaluated in the same small validation cohort. Among 1670 women screened, 34 (2.04%) were identified as SMN1 carriers and 20 (1.20%) as CFTR p.Phe508del carriers. Partner testing identified two carrier couples. Conclusions: The DBS-based multiplex dPCR workflow demonstrated complete analytical concordance with reference methods in this preliminary validation and was implemented in a population screening cohort. Its targeted scope leaves residual carrier risk, requiring appropriate counseling. Larger independent validation and formal throughput and cost assessments are needed before routine implementation.