Single-Tube Multiplex Digital Polymerase Chain Reaction Assay for Molecular Diagnosis and Prediction of Severity of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease characterized by the degeneration of motor neurons and progressive muscle atrophy. Accurate detection of SMN1 and SMN2 copy numbers is essential for SMA diagnosis, carrier screening, disease severity prediction, therapy, and prognosis. However, a method for SMN1 and SMN2 copy number determination that is simultaneously accurate, simple, rapid, multitargeted, and applicable to various samples has not previously been reported. Here, we developed a single-tube multiplex digital polymerase chain reaction (dPCR) assay for simultaneous determination of the copy numbers of SMN1 exons 7 and 8 and SMN2 exons 7 and 8. A total of 317 clinical samples, including peripheral blood, amniotic fluid, chorionic villus, buccal swabs, and dried blood spots, were collected to evaluate the performance of this dPCR-based assay. The test results were accurate for all the clinical samples. Our assay is accurate, rapid, easy to handle, and applicable to many types of samples and uses a small amount of DNA; it is a powerful tool for SMA molecular diagnosis, large-scale screening, and disease severity assessment.