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

被引:28
作者
Gutierrez-Mateo, Cristina [1 ]
Timonen, Anne [2 ]
Vaahtera, Katja [2 ]
Jaakkola, Markku [2 ]
Hougaard, David M. [3 ]
Bybjerg-Grauholm, Jonas [3 ]
Baekvad-Hansen, Marie [3 ]
Adamsen, Dea [3 ]
Filippov, Galina [1 ]
Dallaire, Stephanie [1 ]
Goldfarb, David [1 ]
Schoener, Daniel [1 ]
Wu, Rongcong [1 ]
机构
[1] PerkinElmer, 940 Winter St, Waltham, MA 02451 USA
[2] Wallac Oy, PerkinElmer, Mustionkatu 6, Turku 20750, Finland
[3] Statens Serum Inst, Danish Ctr Neonatal Screening, DK-2300 Copenhagen, Denmark
关键词
Newborn Screening; SCID; SMA; XLA; DBS; real-time PCR; TREC; KREC; SMN1; SEVERE COMBINED IMMUNODEFICIENCY; SPINAL MUSCULAR-ATROPHY; DISEASES; SMN1;
D O I
10.3390/ijns5040039
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
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.
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页数:11
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