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Open AccessArticle

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

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PerkinElmer, 940 Winter St, Waltham, MA 02451, USA
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PerkinElmer, Wallac Oy, Mustionkatu 6, 20750 Turku, Finland
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Danish Center for Neonatal Screening, Statens Serum Institut, 2300 Copenhagen, Denmark
*
Author to whom correspondence should be addressed.
Int. J. Neonatal Screen. 2019, 5(4), 39; https://doi.org/10.3390/ijns5040039
Received: 1 September 2019 / Revised: 25 October 2019 / Accepted: 31 October 2019 / Published: 2 November 2019
(This article belongs to the Special Issue Selected Papers from 11th ISNS European Regional Meeting)
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. View Full-Text
Keywords: Newborn Screening; SCID; SMA; XLA; DBS; real-time PCR; TREC; KREC; SMN1 Newborn Screening; SCID; SMA; XLA; DBS; real-time PCR; TREC; KREC; SMN1
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Gutierrez-Mateo, C.; Timonen, A.; Vaahtera, K.; Jaakkola, M.; Hougaard, D.M.; Bybjerg-Grauholm, J.; Baekvad-Hansen, M.; Adamsen, D.; Filippov, G.; Dallaire, S.; Goldfarb, D.; Schoener, D.; Wu, R. Development of a Multiplex Real-Time PCR Assay for the Newborn Screening of SCID, SMA, and XLA. Int. J. Neonatal Screen. 2019, 5, 39.

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