{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,26]],"date-time":"2025-10-26T21:21:15Z","timestamp":1761513675533},"reference-count":23,"publisher":"Ovid Technologies (Wolters Kluwer Health)","issue":"9","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2016,10]]},"abstract":"<jats:p>Hereditary hemochromatosis is known as the most common genetic disorder among individuals of European genetic background. It is possible to find 2 mutations closely placed in the <jats:italic toggle=\"yes\">HFE<\/jats:italic> gene (H63D and S65C) and this proximity can cause errors when genotyped by real-time polymerase chain reaction (PCR) genotyping assay. The aim of this study was to develop a hydrolysis probe-based PCR assay for detection of the H63D and S65C mutations without interference from on each other. Herein the study involved the standardization of an improvement of the real-time PCR 5\u2032 nuclease assay to detect the desired mutations close placed using a same probe system. The assay analytical properties performances were tested, including the primers selectivity and detection limits. Also, the interexaminer reproducibility and repeatability of assay were estimated in 30 blood samples. Others 153 results of samples were compared with reference method (PCR_RFLP) and the accordance of the results evaluated by Fleiss\u2019 \u03ba method. The results of variation of interexaminer reproducibility and repeatability of assay were not statistically relevant (<jats:italic toggle=\"yes\">P<\/jats:italic>&lt;0.001). The comparison between the 2 methods by Fleiss\u2019 \u03ba analysis showed that 5\u2032 nuclease assay identified the H63D and S65C haplotype as well as the reference method in all 153 tested samples. Our results showed that novel method probe-based real-time PCR were capable to detect 2 adjacent polymorphisms without errors in genotyping.<\/jats:p>","DOI":"10.1097\/pai.0000000000000206","type":"journal-article","created":{"date-parts":[[2015,8,8]],"date-time":"2015-08-08T17:45:20Z","timestamp":1439055920000},"page":"673-678","source":"Crossref","is-referenced-by-count":1,"title":["Novel Method Probe-based Real-Time PCR to Detect 2 Single-Nucleotide Polymorphisms Close to Each Other: HFE Hemochromatosis Gene Model"],"prefix":"10.1097","volume":"24","author":[{"given":"Frederico S. V.","family":"Malta","sequence":"first","affiliation":[{"name":"Departamento de Ginecologia e Obstetr\u00edcia, Faculdade de Medicina, Universidade Federal de Minas Gerais, Av. Prof. Alfredo Balena, Belo Horizonte"},{"name":"Departamento de Pesquisa & Desenvolvimento, Instituto Hermes Pardini, Vespasiano, Minas Gerais, Brazil"}]},{"given":"Zilma N.","family":"Reis","sequence":"additional","affiliation":[{"name":"Departamento de Ginecologia e Obstetr\u00edcia, Faculdade de Medicina, Universidade Federal de Minas Gerais, Av. Prof. Alfredo Balena, Belo Horizonte"}]},{"given":"Ant\u00f4nio C. V.","family":"Cabral","sequence":"additional","affiliation":[{"name":"Departamento de Ginecologia e Obstetr\u00edcia, Faculdade de Medicina, Universidade Federal de Minas Gerais, Av. 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