Evaluation of a Newly Developed Real-time PCR (TaqMan) Assay for Screening Spinal Muscular Atrophy Exon 7 Deletion

Abstract

Background and Objective: Spinal muscular atrophy (SMA) is caused by SMN1 gene mutations, primarily exon 7 deletion. Newborn screening is deemed essential for early diagnosis, but commercial kits like the Eonis™ SCID-SMA Kit are considered costly for resource-limited settings. This study was designed to develop a cost-effective real-time PCR (TaqMan) assay for detecting SMN1 exon 7 deletion and compare its performance with the commercial kit.

Methods: Forty-one dried blood spot (DBS) samples, including 15 EQA-SMAPT quality control samples (9 SMA, 6 non-SMA) and 26 clinical newborn screening samples (7 SMA, 19 non-SMA), were analyzed. The developed assay (β-actin as internal control) and Eonis™ SCID-SMA Kit (RPP30 as internal control) were tested using multiplex real-time PCR. Diagnostic concordance was assessed with Cohen’s Kappa coefficient, Cycle Threshold (Ct) differences with paired t-test (p < 0.05), and costs were compared.

Results: Complete concordance (100%) was observed between the developed assay and Eonis™ kit (Cohen’s Kappa = 1.0, p < 0.001). Ct values > 40 were recorded in SMA samples, indicating SMN1 absence, while mean Ct values of 23.57–25.56 (SD = 0.39–2.07) were obtained in non-SMA samples, confirming SMN1 presence. Significant differences were detected (p < 0.001). No differences were found between β-actin and RPP30 Ct values. The developed assay’s cost was 150 THB/test versus 500 THB for the commercial kit.

Conclusion: The developed assay’s efficacy matches the Eonis™ kit at one-third the cost, ideal for resource-limited settings. Further validation with larger cohorts is recommended.