Reference Measurement Systems in Genetic Testing: Development of DNA-based Reference Materials

Márki-Zay János
Reference Measurement Systems in Genetic Testing: Development of DNA-based Reference Materials.
Doctoral thesis (PhD), University of Szeged.
(2011) (Unpublished)

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Abstract in foreign language

Recent progress in molecular pathology and biotechnology has made DNA-based assays important tools in patient care. DNA tests allow the detection of genetic alterations responsible for inheritable disease conditions, higher risk of developing diseases or altered drug effects. These tests have a high impact on clinical decision-making and thousands of different tests are already in routine use. Thus, quality issues and standardisation of DNA tests are particularly important. Reference materials (RMs) and certified reference materials (CRMs) are recognised as an excellent tool for checking analytical accuracy and are valuable in creating the necessary reference points in the development of comprehensive measurement systems. (C)RMs are used for method development and validation, calibration, statistical quality control within a laboratory and to assess the performance of laboratories in proficiency testing schemes. At present, the lack of (C)RMs for molecular genetic tests means that there are limitations in benchmarking against which a laboratory can judge the performance of its assays. Thus, the need for appropriate (C)RMs became increasingly urgent. In the frame of these projects, prototypes for different types of genetic RMs were processed for inherited diseases and genetic risk factors, such as hereditary hemochromatosis, fragile X syndrome, cystic fibrosis, the factor V Leiden (G1691A) mutation and Factor II (prothrombin) G20210A variant. These prototype RMs were evaluated for their suitability and different possibilities for the preservation and packaging of purified DNA were investigated in order to improve the stability and recovery rate of DNA-based RMs. Utilizing these results and experiences, a set of plasmid-type RMs for the analysis of the human prothrombin gene G20210A mutation were developed and produced. These candidate CRMs were characterised using quantitative real-time PCR techniques for homogeneity, stability, sequence identity and fitness for purpose. The material is homogeneous and stable at -20 °C. The sequence of the insert was confirmed by DNA sequence analysis. Each vial contains an indicative volume of 50 μL corresponding to approximately 1 ng of plasmid DNA. This series of plasmid CRMs was certified according to the ISO Guides 30−35 and is now available from the IRMM to support the validation and the harmonisation of polymerase chain reaction (PCR)-based methods used for detection of the G20210A mutation in the human prothrombin gene. The CRMs IRMM/IFCC-490 (G20210/G20210 wild-type), IRMM/IFCC-491 (20210A/20210A homozygous mutant) and IRMM/IFCC-492 (G20210/20210A heterozygous mutant) have been the first clinical genetic CRMs introduced worldwide. In addition, these reference plasmids were used also for the production of quality control materials for rare sequence variants to carry out a European proficiency study for the assessment of the competence of clinical laboratories to recognize and correctly interpret unusual genotyping results caused by rare SNPs. Our experiences and knowledge gained from these projects can be used in the development and production of further DNA-based CRMs for any molecular genetic test.

Item Type: Thesis (Doctoral thesis (PhD))
Creators: Márki-Zay János
Hungarian title: Referencia mérési rendszerek a genetikai diagnosztikában: DNS-alapú referencia anyagok fejlesztése
Position, academic title, institution
MTMT author ID
Klein Christoph L.
tanszékvezető egyetemi tanár, Phd, DSc, Scientific Officer, PhD, MBA, European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Geel, Belgium
Dux László
tanszékvezető egyetemi tanár, Phd, DSc, SZTE ÁOK Biokémia Intézet
Subjects: 03. Medical and health sciences > 03.01. Basic medicine
Divisions: Doctoral School of Multidisciplinary Medical Sciences
Discipline: Medicine > Theoretical Medicine
Medicine > Clinical Medicine
Language: English
Date: 2011. November 25.
Item ID: 1118
MTMT identifier of the thesis: 1930875
Date Deposited: 2011. Nov. 05. 08:08
Last Modified: 2019. Nov. 26. 14:54
Depository no.: B 4971
Defence/Citable status: Defended.

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