{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T02:48:49Z","timestamp":1780541329919,"version":"3.54.1"},"reference-count":47,"publisher":"Public Library of Science (PLoS)","issue":"12","license":[{"start":{"date-parts":[[2022,12,1]],"date-time":"2022-12-01T00:00:00Z","timestamp":1669852800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Semmelweis Science and Innovation Fund","award":["STIA-KF-17"],"award-info":[{"award-number":["STIA-KF-17"]}]},{"DOI":"10.13039\/501100003549","name":"Hungarian Scientific Research Fund","doi-asserted-by":"publisher","award":["K125231"],"award-info":[{"award-number":["K125231"]}],"id":[{"id":"10.13039\/501100003549","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Thematic Excellence Program Health Subprogram (MOLORKIV) of the Ministry for Innovation and Technology in Hungary"},{"name":"Janos Bolyai Research Scholarship"},{"name":"New National Excellence Program of the Ministry for Innovation and Technology","award":["UNKP-19-4"],"award-info":[{"award-number":["UNKP-19-4"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Quantitative PCR (qPCR) is used for the determination of gene copy number (GCN). GCNs contribute to human disorders, and characterize copy number variation (CNV). The single laboratory method validations of duplex qPCR assays with hydrolysis probes on <jats:italic>CYP21A1P<\/jats:italic> and <jats:italic>CYP21A2<\/jats:italic> genes, residing a CNV (RCCX CNV) and related to congenital adrenal hyperplasia, were performed using 46 human genomic DNA samples. We also performed the verifications on 5 qPCR assays for the genetic elements of RCCX CNV; <jats:italic>C4A<\/jats:italic>, <jats:italic>C4B<\/jats:italic>, CNV breakpoint, HERV-K(C4) CNV deletion and insertion alleles. Precision of each qPCR assay was under 1.01 CV%. Accuracy (relative error) ranged from 4.96\u00b14.08% to 9.91\u00b18.93%. Accuracy was not tightly linked to precision, but was significantly correlated with the efficiency of normalization using the <jats:italic>RPPH1<\/jats:italic> internal reference gene (Spearman\u2019s \u03c1: 0.793\u20130.940, p&gt;0.0001), ambiguity (\u03c1 = 0.671, p = 0.029) and misclassification (\u03c1 = 0.769, p = 0.009). A strong genomic matrix effect was observed, and target-singleplex (one target gene in one assay) qPCR was able to appropriately differentiate 2 GCN from 3 GCN at best. The analysis of all GCNs from the 7 qPCR assays using a multiplex approach increased the resolution of differentiation, and produced 98% of GCNs unambiguously, and all of which were in 100% concordance with GCNs measured by Southern blot, MLPA and aCGH. We conclude that the use of an internal (in one assay with the target gene) reference gene, the use of allele-specific primers or probes, and the multiplex approach (in one assay or different assays) are crucial for GCN determination using qPCR or other methods.<\/jats:p>","DOI":"10.1371\/journal.pone.0277299","type":"journal-article","created":{"date-parts":[[2022,12,1]],"date-time":"2022-12-01T19:43:14Z","timestamp":1669923794000},"page":"e0277299","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":7,"title":["Quantitative PCR from human genomic DNA: The determination of gene copy numbers for congenital adrenal hyperplasia and RCCX copy number variation"],"prefix":"10.1371","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0909-5232","authenticated-orcid":true,"given":"M\u00e1rton","family":"Doleschall","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ott\u00f3","family":"Darvasi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5990-4889","authenticated-orcid":true,"given":"Zolt\u00e1n","family":"Herold","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zolt\u00e1n","family":"Doleschall","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0248-0007","authenticated-orcid":true,"given":"G\u00e1bor","family":"Nyir\u0151","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anik\u00f3","family":"Somogyi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2192-554X","authenticated-orcid":true,"given":"P\u00e9ter","family":"Igaz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Attila","family":"Pat\u00f3cs","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2022,12,1]]},"reference":[{"key":"pone.0277299.ref001","doi-asserted-by":"crossref","first-page":"1158","DOI":"10.1093\/infdis\/158.6.1158","article-title":"Enzymatic gene amplification: qualitative and quantitative methods for detecting proviral DNA amplified in vitro","volume":"158","author":"MA Abbott","year":"1988","journal-title":"J Infect Dis"},{"key":"pone.0277299.ref002","doi-asserted-by":"crossref","first-page":"619","DOI":"10.2144\/000112776","article-title":"Twenty-five years of quantitative PCR for gene expression analysis","volume":"44","author":"HD VanGuilder","year":"2008","journal-title":"Biotechniques"},{"key":"pone.0277299.ref003","doi-asserted-by":"crossref","first-page":"752","DOI":"10.1093\/clinchem\/43.5.752","article-title":"Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erbB-2 oncogene amplification","volume":"43","author":"S Gelmini","year":"1997","journal-title":"Clin Chem"},{"key":"pone.0277299.ref004","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1111\/j.1399-0004.2009.01342.x","article-title":"Gene copy number variation and common human disease","volume":"77","author":"M Fanciulli","year":"2010","journal-title":"Clin Genet"},{"key":"pone.0277299.ref005","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1016\/j.mcp.2006.03.003","article-title":"Evaluation of minor groove binding probe and Taqman probe PCR assays: Influence of mismatches and template complexity on quantification","volume":"20","author":"Y Yao","year":"2006","journal-title":"Mol Cell Probes"},{"key":"pone.0277299.ref006","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1016\/j.cell.2011.09.008","article-title":"Clan genomics and the complex architecture of human disease","volume":"147","author":"JR Lupski","year":"2011","journal-title":"Cell"},{"key":"pone.0277299.ref007","doi-asserted-by":"crossref","first-page":"660","DOI":"10.1126\/science.aaa0355","article-title":"Human genomics. 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