Development of a Multiplex Real-Time PCR Assay for the Newborn Screening of SCID, SMA, and XLA.

Numerous studies have shown evidence supporting the benefits of universal newborn screening for primary immunodeficiencies (PID) and for Spinal Muscular Atrophy (SMA). We have developed a four-plex, real-time PCR assay to screen for Severe Combined Immune Deficiencies (SCID), X-linked agammaglobulinemia (XLA), and SMA in DNA extracted from a single 3.2 mm punch of a dried blood spot (DBS). A simple, high-throughput, semi-automated DNA extraction method was developed for a Janus liquid handler that can process 384 DBS punches in four 96-well plates in just over one hour with sample tracking capability. The PCR assay identifies the absence of exon 7 in the SMN1 gene, while simultaneously evaluating the copy number of T-cell receptor excision circles (TREC) and Kappa-deleting recombination excision circles (KREC) molecules. Additionally, the amplification of a reference gene, RPP30 , was included in the assay as a quality/quantity indicator of DNA isolated from the DBS. The assay performance was demonstrated on over 3000 DNA samples isolated from punches of putative normal newborn DBS. The reliability and analytical accuracy were further evaluated using DBS controls, and contrived and confirmed positive samples. The results from this study demonstrate the potential of future molecular DBS assays, and highlight how a multiplex assay could benefit newborn screening programs.
Competing Interests: Conflicts of InterestC.G-M., A.T., K.V., M.J., G.F., S.D., D.G., D.S. and R.W. are PerkinElmer employees. Authors representing PerkinElmer had a role in designing the study, in collection, analyses, and interpretation of the data, in the writing of the manuscript, and in the decision to publish the results. The other authors declare no conflict of interest.
(© 2019 by the authors.)