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Development of a thermostabilised PCR-based detection kit for pathogenic familial hypercholesterolaemia variants in Malaysia / Norhidayah Rosman

Familial hypercholesterolaemia (FH) is an inherited disease that causes an elevation of plasma low-density lipoprotein cholesterol (LDL-C) level, leading to increased risk of premature coronary artery disease. Next-generation sequencing (NGS) is currently used to detect FH variants molecularly among...

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Main Author: Rosman, Norhidayah
Format: Thesis
Language:English
Published: 2022
Subjects:
Diseases of the circulatory (Cardiovascular) system
Individual drugs and other agents
Online Access:https://ir.uitm.edu.my/id/eprint/77969/1/77969.pdf
https://ir.uitm.edu.my/id/eprint/77969/
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spelling my.uitm.ir.779692023-05-29T07:39:44Z https://ir.uitm.edu.my/id/eprint/77969/ Development of a thermostabilised PCR-based detection kit for pathogenic familial hypercholesterolaemia variants in Malaysia / Norhidayah Rosman Rosman, Norhidayah Diseases of the circulatory (Cardiovascular) system Individual drugs and other agents Familial hypercholesterolaemia (FH) is an inherited disease that causes an elevation of plasma low-density lipoprotein cholesterol (LDL-C) level, leading to increased risk of premature coronary artery disease. Next-generation sequencing (NGS) is currently used to detect FH variants molecularly among patients. However, this method is expensive, laborious, and time-consuming. Thus, a simpler method using a tetra-primer samplification refractory mutation system (T-ARMS) PCR was developed for detection of 10 most common pathogenic variants in Malaysia. The kit was designed to detect 9pathogenic variants of the LDLR gene and 1 APOB gene pathogenic variant. The sevariants were selected by analysing their pathogenicity and their frequency among molecularly confirmed FH cases from previous published and unpublished data. The ratio of inner and outer primers’ concentration of each variant and the annealing temperature were optimised to achieve optimal results. The optimised PCR was then evaluated with 154 clinical samples to determine the diagnostic performance of this kit.Limit of detection (LoD) was performed using synthetic DNA targets as well as extracted patient DNA. The diagnostic performance of the kit showed 100% for sensitivity, specificity PPV, NPV and accuracy. The LoD was 1.0X10-2 ng for syntheticDNA and 10.0 ng for the extracted DNA from FH and non-FH patients. A prototype was developed by using a 96-well PCR plate with lyophilised primers of each variant dispensed into different wells. The stability of the prototype was analysed using the Q10accelerated aging method. This method showed the kit was stable at room temperature for up to three months. This thermostabilised T-ARMS PCR prototype provides a simple-to-use kit that can be performed using a simple PCR thermocycler for the rapid screening of pathogenic FH variants. It may also be useful for molecular confirmation of FH zygosity in the regional Asian countries. Easy identification of pathogenic FHvariants will allow prompt and early intervention, thus reducing the risk of coronary artery disease among the population. 2022 Thesis NonPeerReviewed text en https://ir.uitm.edu.my/id/eprint/77969/1/77969.pdf Development of a thermostabilised PCR-based detection kit for pathogenic familial hypercholesterolaemia variants in Malaysia / Norhidayah Rosman. (2022) Masters thesis, thesis, Universiti Teknologi MARA (UiTM). <http://terminalib.uitm.edu.my/77969.pdf>
institution Universiti Teknologi Mara
building Tun Abdul Razak Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Teknologi Mara
content_source UiTM Institutional Repository
url_provider http://ir.uitm.edu.my/
language English
topic Diseases of the circulatory (Cardiovascular) system
Individual drugs and other agents
spellingShingle Diseases of the circulatory (Cardiovascular) system
Individual drugs and other agents
Rosman, Norhidayah
Development of a thermostabilised PCR-based detection kit for pathogenic familial hypercholesterolaemia variants in Malaysia / Norhidayah Rosman
description Familial hypercholesterolaemia (FH) is an inherited disease that causes an elevation of plasma low-density lipoprotein cholesterol (LDL-C) level, leading to increased risk of premature coronary artery disease. Next-generation sequencing (NGS) is currently used to detect FH variants molecularly among patients. However, this method is expensive, laborious, and time-consuming. Thus, a simpler method using a tetra-primer samplification refractory mutation system (T-ARMS) PCR was developed for detection of 10 most common pathogenic variants in Malaysia. The kit was designed to detect 9pathogenic variants of the LDLR gene and 1 APOB gene pathogenic variant. The sevariants were selected by analysing their pathogenicity and their frequency among molecularly confirmed FH cases from previous published and unpublished data. The ratio of inner and outer primers’ concentration of each variant and the annealing temperature were optimised to achieve optimal results. The optimised PCR was then evaluated with 154 clinical samples to determine the diagnostic performance of this kit.Limit of detection (LoD) was performed using synthetic DNA targets as well as extracted patient DNA. The diagnostic performance of the kit showed 100% for sensitivity, specificity PPV, NPV and accuracy. The LoD was 1.0X10-2 ng for syntheticDNA and 10.0 ng for the extracted DNA from FH and non-FH patients. A prototype was developed by using a 96-well PCR plate with lyophilised primers of each variant dispensed into different wells. The stability of the prototype was analysed using the Q10accelerated aging method. This method showed the kit was stable at room temperature for up to three months. This thermostabilised T-ARMS PCR prototype provides a simple-to-use kit that can be performed using a simple PCR thermocycler for the rapid screening of pathogenic FH variants. It may also be useful for molecular confirmation of FH zygosity in the regional Asian countries. Easy identification of pathogenic FHvariants will allow prompt and early intervention, thus reducing the risk of coronary artery disease among the population.
format Thesis
author Rosman, Norhidayah
author_facet Rosman, Norhidayah
author_sort Rosman, Norhidayah
title Development of a thermostabilised PCR-based detection kit for pathogenic familial hypercholesterolaemia variants in Malaysia / Norhidayah Rosman
title_short Development of a thermostabilised PCR-based detection kit for pathogenic familial hypercholesterolaemia variants in Malaysia / Norhidayah Rosman
title_full Development of a thermostabilised PCR-based detection kit for pathogenic familial hypercholesterolaemia variants in Malaysia / Norhidayah Rosman
title_fullStr Development of a thermostabilised PCR-based detection kit for pathogenic familial hypercholesterolaemia variants in Malaysia / Norhidayah Rosman
title_full_unstemmed Development of a thermostabilised PCR-based detection kit for pathogenic familial hypercholesterolaemia variants in Malaysia / Norhidayah Rosman
title_sort development of a thermostabilised pcr-based detection kit for pathogenic familial hypercholesterolaemia variants in malaysia / norhidayah rosman
publishDate 2022
url https://ir.uitm.edu.my/id/eprint/77969/1/77969.pdf
https://ir.uitm.edu.my/id/eprint/77969/
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score 13.211869

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