Novel Method Probe-based Real-Time PCR to Detect 2 Single-Nucleotide Polymorphisms Close to Each Other: HFE Hemochromatosis Gene Model

摘要

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 HFE 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' 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 interexa-miner 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' k method. The results of variation of interexaminer reproducibility and repeatability of assay were not statistically relevant (P < 0.001). The comparison between the 2 methods by Fleiss' k analysis showed that 5' 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.