{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:49:43Z","timestamp":1787028583057,"version":"build-2736575974"},"reference-count":32,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2019,11,2]],"date-time":"2019-11-02T00:00:00Z","timestamp":1572652800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJNS"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/ijns5040039","type":"journal-article","created":{"date-parts":[[2019,11,4]],"date-time":"2019-11-04T04:13:08Z","timestamp":1572840788000},"page":"39","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":38,"title":["Development of a Multiplex Real-Time PCR Assay for the Newborn Screening of SCID, SMA, and XLA"],"prefix":"10.3390","volume":"5","author":[{"given":"Cristina","family":"Gutierrez-Mateo","sequence":"first","affiliation":[{"name":"PerkinElmer, 940 Winter St, Waltham, MA 02451, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anne","family":"Timonen","sequence":"additional","affiliation":[{"name":"PerkinElmer, Wallac Oy, Mustionkatu 6, 20750 Turku, Finland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katja","family":"Vaahtera","sequence":"additional","affiliation":[{"name":"PerkinElmer, Wallac Oy, Mustionkatu 6, 20750 Turku, Finland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Markku","family":"Jaakkola","sequence":"additional","affiliation":[{"name":"PerkinElmer, Wallac Oy, Mustionkatu 6, 20750 Turku, Finland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David M","family":"Hougaard","sequence":"additional","affiliation":[{"name":"Danish Center for Neonatal Screening, Statens Serum Institut, 2300 Copenhagen, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonas","family":"Bybjerg-Grauholm","sequence":"additional","affiliation":[{"name":"Danish Center for Neonatal Screening, Statens Serum Institut, 2300 Copenhagen, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5881-1776","authenticated-orcid":false,"given":"Marie","family":"Baekvad-Hansen","sequence":"additional","affiliation":[{"name":"Danish Center for Neonatal Screening, Statens Serum Institut, 2300 Copenhagen, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dea","family":"Adamsen","sequence":"additional","affiliation":[{"name":"Danish Center for Neonatal Screening, Statens Serum Institut, 2300 Copenhagen, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Galina","family":"Filippov","sequence":"additional","affiliation":[{"name":"PerkinElmer, 940 Winter St, Waltham, MA 02451, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephanie","family":"Dallaire","sequence":"additional","affiliation":[{"name":"PerkinElmer, 940 Winter St, Waltham, MA 02451, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0143-1924","authenticated-orcid":false,"given":"David","family":"Goldfarb","sequence":"additional","affiliation":[{"name":"PerkinElmer, 940 Winter St, Waltham, MA 02451, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Schoener","sequence":"additional","affiliation":[{"name":"PerkinElmer, 940 Winter St, Waltham, MA 02451, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rongcong","family":"Wu","sequence":"additional","affiliation":[{"name":"PerkinElmer, 940 Winter St, Waltham, MA 02451, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2019,11,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1038\/gim.2012.114","article-title":"An empirical estimate of carrier frequencies for 400+ causal Mendelian variants: Results from an ethnically diverse clinical sample of 23,453 individuals","volume":"15","author":"Lazarin","year":"2013","journal-title":"Genet. Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1723","DOI":"10.1056\/NEJMoa1702752","article-title":"Nusinersen versus Sham Control in Infantile-Onset Spinal Muscular Atrophy","volume":"377","author":"Finkel","year":"2017","journal-title":"N. Engl. J. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1713","DOI":"10.1056\/NEJMoa1706198","article-title":"Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy","volume":"377","author":"Mendell","year":"2017","journal-title":"N. Engl. J. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/j.pediatrneurol.2019.05.005","article-title":"Impact of Age and Motor Function in a Phase 1\/2A Study of Infants with SMA Type 1 Receiving Single-Dose Gene Replacement Therapy","volume":"98","author":"Lowes","year":"2019","journal-title":"Pediatr. Neurol."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"American College of Medical Genetics Newborn Screening Expert Group (2006). Newborn Screening: Towards a Uniform Screening Panel and System. Genet. Med., 117, S296\u2013S307.","DOI":"10.1542\/peds.2005-2633I"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"American College of Medical Genetics Newborn Screening Expert Group (2006). Naming and Counting Disorders (Conditions) Included in Newborn Screening Panels. Pediatrics, 117, S308\u2013S314.","DOI":"10.1542\/peds.2005-2633J"},{"key":"ref_7","unstructured":"(2019, November 01). House Passes AACC Backed Newborn Screening Measure. Available online: https:\/\/www.aacc.org\/."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"522","DOI":"10.1016\/j.jaci.2009.04.007","article-title":"Development of a routine newborn screening protocol for severe combined immunodeficiency","volume":"124","author":"Baker","year":"2009","journal-title":"J. Allergy Clin. Immunol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1016\/j.jaci.2004.10.012","article-title":"Development of population-based newborn screening for severe combined immunodeficiency","volume":"115","author":"Chan","year":"2005","journal-title":"J. Allergy Clin. Immunol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"729","DOI":"10.1001\/jama.2014.9132","article-title":"Newborn screening for severe combined immunodeficiency in 11 screening programs in the United States","volume":"312","author":"Kwan","year":"2014","journal-title":"JAMA"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"226","DOI":"10.1016\/j.jaci.2014.04.026","article-title":"Combined newborn screening for familial hemophagocytic lymphohistiocytosis and severe T- and B-cell immunodeficiencies","volume":"134","author":"Borte","year":"2014","journal-title":"J. Allergy Clin. Immunol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"12699","DOI":"10.1111\/sji.12699","article-title":"Newborn screening using TREC\/KREC assay for severe T and B cell lymphopenia in Iran","volume":"26","author":"Nourizadeh","year":"2018","journal-title":"Scand. J. Immunol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1007\/s10875-017-0455-x","article-title":"Newborn Screening for Primary Immunodeficiency Diseases: History, Current and Future Practice","volume":"38","author":"King","year":"2018","journal-title":"J. Clin. Immunol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"434","DOI":"10.1056\/NEJMoa1401177","article-title":"Transplantation outcomes for severe combined immunodeficiency, 2000\u20132009","volume":"371","author":"Pai","year":"2014","journal-title":"N. Engl. J. Med."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1006\/clim.2002.5241","article-title":"Clinical, immunological, and molecular analysis in a large cohort of patients with X-linked agammaglobulinemia: An Italian multicenter study","volume":"104","author":"Plebani","year":"2002","journal-title":"Clin. Immunol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1608","DOI":"10.1002\/ajmg.a.33474","article-title":"Newborn and carrier screening for spinal muscular atrophy","volume":"152A","author":"Prior","year":"2010","journal-title":"Am. J. Med. Genet. A"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"412","DOI":"10.1373\/clinchem.2014.231019","article-title":"Newborn blood spot screening test using multiplexed real-time PCR to simultaneously screen for spinal muscular atrophy and severe combined immunodeficiency","volume":"61","author":"Taylor","year":"2015","journal-title":"Clin. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1186\/1479-5876-11-188","article-title":"Utilization of TREC and KREC quantification for the monitoring of early T- and B-cell neogenesis in adult patients after allogeneic hematopoietic stem cell transplantation","volume":"11","author":"Mensen","year":"2013","journal-title":"J. Transl. Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1016\/j.clinbiochem.2011.10.019","article-title":"Spinal muscular atrophy: Clinical validation of a single-tube multiplex real time PCR assay for determination of SMN1 and SMN2 copy numbers","volume":"45","author":"Maranda","year":"2012","journal-title":"Clin. Biochem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1111\/imr.12729","article-title":"Newborn screening for severe combined immunodeficiency and T-cell lymphopenia","volume":"287","author":"Puck","year":"2019","journal-title":"Immunol. Rev."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1111\/pai.12501","article-title":"Prospective neonatal screening for severe T- and B-lymphocyte deficiencies in Seville","volume":"27","author":"Olbrich","year":"2016","journal-title":"Pediatr. Allergy Immunol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1007\/s12519-019-00242-6","article-title":"Progress in treatment and newborn screening for Duchenne muscular dystrophy and spinal muscular atrophy","volume":"15","author":"Ke","year":"2019","journal-title":"World J. Pediatr."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1177","DOI":"10.1093\/hmg\/8.7.1177","article-title":"A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2","volume":"8","author":"Monani","year":"1999","journal-title":"Hum. Mol. Genet."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.jpeds.2017.06.042","article-title":"Presymptomatic Diagnosis of Spinal Muscular Atrophy Through Newborn Screening","volume":"190","author":"Chien","year":"2017","journal-title":"J. Pediatr."},{"key":"ref_25","first-page":"1057","article-title":"Hybrid survival motor neuron genes in patients with autosomal recessive spinal muscular atrophy: New insights into molecular mechanisms responsible for the disease","volume":"59","author":"Hahnen","year":"1996","journal-title":"Am. J. Hum. Genet."},{"key":"ref_26","unstructured":"The Evidence-based Review Group (2019, August 21). Evidence-Based Review of Newborn Screening for Spinal Muscular Atrophy (SMA): Final Report (v5.2). Prepared for: Maternal and Child Health Bureau, Available online: https:\/\/www.hrsa.gov\/sites\/default\/files\/hrsa\/advisory-committees\/heritable-disorders\/reports-recommendations\/sma-final-report.pdf."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1016\/j.nmd.2019.02.003","article-title":"Newborn screening for SMA in Southern Belgium","volume":"29","author":"Boemer","year":"2019","journal-title":"Neuromuscul. Disord."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1097\/MOH.0b013e32835a9130","article-title":"Guidelines for newborn screening of primary immunodeficiency diseases","volume":"20","author":"Borte","year":"2013","journal-title":"Curr. Opin. Hematol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1007\/s10875-016-0347-5","article-title":"Newborn screening for severe primary immunodeficiency diseases in Sweden-a 2-year pilot TREC and KREC screening study","volume":"37","author":"Barbaro","year":"2017","journal-title":"J. Clin. Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2552","DOI":"10.1182\/blood-2011-08-371021","article-title":"Neonatal screening for severe primary immunodeficiency diseases using high-throughput triplex real-time PCR","volume":"119","author":"Borte","year":"2012","journal-title":"Blood"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1466","DOI":"10.1373\/clinchem.2010.144915","article-title":"High-throughput multiplexed T-cell-receptor excision circle quantitative PCR assay with internal controls for detection of severe combined immunodeficiency in population-based newborn screening","volume":"56","author":"Wilkey","year":"2010","journal-title":"Clin. Chem."},{"key":"ref_32","unstructured":"EnLite\u2122 Neonatal TREC system (2013). EnLite\u2122 Neonatal TREC Kit 3401-0010. Instructions for Use, PerkinElmer Catalogue."}],"container-title":["International Journal of Neonatal Screening"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2409-515X\/5\/4\/39\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T13:31:29Z","timestamp":1760189489000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2409-515X\/5\/4\/39"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,11,2]]},"references-count":32,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2019,12]]}},"alternative-id":["ijns5040039"],"URL":"https:\/\/doi.org\/10.3390\/ijns5040039","relation":{},"ISSN":["2409-515X"],"issn-type":[{"value":"2409-515X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,11,2]]}}}