Development of an Optimized Tetra-Amplification Refractory Mutation System PCR for Detection of 12 Pathogenic Familial Hypercholesterolemia Variants in the Asian Population.

Autor:	Rosman N; Institute of Pathology, Laboratory and Forensic Medicine (I-PPerForM), Universiti Teknologi MARA, Sungai Buloh Campus, Selangor, Malaysia; Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh Campus, Selangor, Malaysia., Nawawi HM; Institute of Pathology, Laboratory and Forensic Medicine (I-PPerForM), Universiti Teknologi MARA, Sungai Buloh Campus, Selangor, Malaysia; Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh Campus, Selangor, Malaysia., Al-Khateeb A; Institute of Pathology, Laboratory and Forensic Medicine (I-PPerForM), Universiti Teknologi MARA, Sungai Buloh Campus, Selangor, Malaysia; Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh Campus, Selangor, Malaysia., Chua YA; Institute of Pathology, Laboratory and Forensic Medicine (I-PPerForM), Universiti Teknologi MARA, Sungai Buloh Campus, Selangor, Malaysia., Chua AL; Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh Campus, Selangor, Malaysia. Electronic address: anglim@uitm.edu.my.
Jazyk:	angličtina
Zdroj:	The Journal of molecular diagnostics : JMD [J Mol Diagn] 2022 Feb; Vol. 24 (2), pp. 120-130.
DOI:	10.1016/j.jmoldx.2021.10.006
Abstrakt:	Early detection of genetic diseases such as familial hypercholesterolemia (FH), and the confirmation of related pathogenic variants, are crucial in reducing the risk for premature coronary artery disease. Currently, next-generation sequencing is used for detecting FH-related candidate genes but is expensive and time-consuming. There is a lack of kits suitable for the detection of the common FH-related variants in the Asia-Pacific region. Thus, this study addressed that need with the development of an optimized tetra-amplification mutation system (T-ARMS) PCR-based assay for the detection of 12 pathogenic variants of FH in the Asian population. The two important parameters for T-ARMS PCR assay performance-annealing temperature and the ratio of outer/inner primer concentrations-were optimized in this study. The optimal annealing temperature of all 12 T-ARMS PCR reactions was 64.6°C. The ideal ratios of outer/inner primer concentrations with each pathogenic variant were: A1, 1:2; A2, 1:4; L1, 1:10; L2, 1:1; L3, 1:2; L4, 1:8; L5, 1:1; L6, 1:2; L7, 1:8; L8, 1:8; L9, 1:2; and L10, 1:8. The lowest limit of detection using DNA extracted from patients was 0.1 ng. The present article highlights the beneficial findings on T-ARMS PCR as part of the development of a PCR-based detection kit for use in detecting FH in economically developing countries in Asia with a greater prevalence of FH. (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu