开发基于 DNA 的侧流检测法,用于检测家族性高胆固醇血症的 LDLR 基因突变。

IF 1.1 Q4 MEDICINE, RESEARCH & EXPERIMENTAL
Malaysian Journal of Medical Sciences Pub Date : 2024-06-01 Epub Date: 2024-06-27 DOI:10.21315/mjms2024.31.3.6
Lina Khalida Saidi, Zam Zureena Md Rani, Siti Aishah Sulaiman, Rahman Jamal, Aziah Ismail, Anis Amirah Alim, Sharipah Nadzirah Syed Ahmad Ayob, Chang Fu Dee, Azrul Azlan Hamzah, Nor Azian Abdul Murad
{"title":"开发基于 DNA 的侧流检测法,用于检测家族性高胆固醇血症的 LDLR 基因突变。","authors":"Lina Khalida Saidi, Zam Zureena Md Rani, Siti Aishah Sulaiman, Rahman Jamal, Aziah Ismail, Anis Amirah Alim, Sharipah Nadzirah Syed Ahmad Ayob, Chang Fu Dee, Azrul Azlan Hamzah, Nor Azian Abdul Murad","doi":"10.21315/mjms2024.31.3.6","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>The techniques for detecting single nucleotide polymorphisms (SNP) require lengthy and complex experimental procedures and expensive instruments that may only be available in some laboratories. Thus, a deoxyribonucleic acid (DNA)-based lateral flow assay (LFA) was developed as a point-of-care test (POCT) diagnostic tool for genotyping. In this study, single nucleotide variation (E101K) in the low-density lipoprotein receptor <i>(LDLR)</i> gene leading to familial hypercholesterolemia (FH) was chosen as a model.</p><p><strong>Methods: </strong>Hypercholesterolemic individuals (<i>n</i> = 103) were selected from the Malaysian Cohort project (UKM Medical Molecular Biology Institute) while the control samples were selected from the Biobank (UKM Medical Molecular Biology Institute). The DNA samples were isolated from whole blood. Polymerase chain reaction (PCR) amplification process was performed using bifunctional labelled primers specifically designed to correspond to the variant that differentiates wild-type and mutant DNA for visual detection on LFA. The variant was confirmed using Sanger sequencing, and the sensitivity and specificity of the LFA detection method were validated using the Agena MassARRAY<sup>®</sup> technique.</p><p><strong>Results: </strong>Out of 103 hypercholesterolemic individuals, 5 individuals (4.8%) tested positive for E101K, <i>LDLR</i> mutation and the rest, including healthy control individuals, tested negative. This result was concordant with Sanger sequencing and Agena MassARRAY<sup>®</sup>. These five individuals could be classified as Definite FH, as the DNA diagnosis was confirmed. The sensitivity and specificity of the variant detection by LFA is 100% compared to results using the genotyping method using Agena MassARRAY<sup>®</sup>.</p><p><strong>Conclusion: </strong>The developed LFA can potentially be used in the POC setting for detecting the E101K variant in the <i>LDLR</i> gene. This LFA can also be used to screen family members with E101K variant in the <i>LDLR</i> gene and is applicable for other SNP's detection.</p>","PeriodicalId":47388,"journal":{"name":"Malaysian Journal of Medical Sciences","volume":null,"pages":null},"PeriodicalIF":1.1000,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11229576/pdf/","citationCount":"0","resultStr":"{\"title\":\"Development of DNA-Based Lateral Flow Assay for Detection of LDLR Gene Mutation for Familial Hypercholesterolemia.\",\"authors\":\"Lina Khalida Saidi, Zam Zureena Md Rani, Siti Aishah Sulaiman, Rahman Jamal, Aziah Ismail, Anis Amirah Alim, Sharipah Nadzirah Syed Ahmad Ayob, Chang Fu Dee, Azrul Azlan Hamzah, Nor Azian Abdul Murad\",\"doi\":\"10.21315/mjms2024.31.3.6\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>The techniques for detecting single nucleotide polymorphisms (SNP) require lengthy and complex experimental procedures and expensive instruments that may only be available in some laboratories. Thus, a deoxyribonucleic acid (DNA)-based lateral flow assay (LFA) was developed as a point-of-care test (POCT) diagnostic tool for genotyping. In this study, single nucleotide variation (E101K) in the low-density lipoprotein receptor <i>(LDLR)</i> gene leading to familial hypercholesterolemia (FH) was chosen as a model.</p><p><strong>Methods: </strong>Hypercholesterolemic individuals (<i>n</i> = 103) were selected from the Malaysian Cohort project (UKM Medical Molecular Biology Institute) while the control samples were selected from the Biobank (UKM Medical Molecular Biology Institute). The DNA samples were isolated from whole blood. Polymerase chain reaction (PCR) amplification process was performed using bifunctional labelled primers specifically designed to correspond to the variant that differentiates wild-type and mutant DNA for visual detection on LFA. The variant was confirmed using Sanger sequencing, and the sensitivity and specificity of the LFA detection method were validated using the Agena MassARRAY<sup>®</sup> technique.</p><p><strong>Results: </strong>Out of 103 hypercholesterolemic individuals, 5 individuals (4.8%) tested positive for E101K, <i>LDLR</i> mutation and the rest, including healthy control individuals, tested negative. This result was concordant with Sanger sequencing and Agena MassARRAY<sup>®</sup>. These five individuals could be classified as Definite FH, as the DNA diagnosis was confirmed. The sensitivity and specificity of the variant detection by LFA is 100% compared to results using the genotyping method using Agena MassARRAY<sup>®</sup>.</p><p><strong>Conclusion: </strong>The developed LFA can potentially be used in the POC setting for detecting the E101K variant in the <i>LDLR</i> gene. This LFA can also be used to screen family members with E101K variant in the <i>LDLR</i> gene and is applicable for other SNP's detection.</p>\",\"PeriodicalId\":47388,\"journal\":{\"name\":\"Malaysian Journal of Medical Sciences\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.1000,\"publicationDate\":\"2024-06-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11229576/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Malaysian Journal of Medical Sciences\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.21315/mjms2024.31.3.6\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/6/27 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q4\",\"JCRName\":\"MEDICINE, RESEARCH & EXPERIMENTAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Malaysian Journal of Medical Sciences","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.21315/mjms2024.31.3.6","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/6/27 0:00:00","PubModel":"Epub","JCR":"Q4","JCRName":"MEDICINE, RESEARCH & EXPERIMENTAL","Score":null,"Total":0}
引用次数: 0

摘要

背景:检测单核苷酸多态性(SNP)的技术需要冗长而复杂的实验程序和昂贵的仪器,而这些仪器可能只有某些实验室才有。因此,人们开发了一种基于脱氧核糖核酸(DNA)的侧流检测法(LFA),作为一种用于基因分型的床旁检测(POCT)诊断工具。方法:高胆固醇血症患者(n = 103)选自马来西亚队列项目(UKM 医学分子生物学研究所),对照样本选自生物库(UKM 医学分子生物学研究所)。DNA 样本从全血中分离。聚合酶链反应(PCR)扩增过程中使用了双功能标记引物,该引物专门设计用于区分野生型和突变型 DNA 的变异体,以便在 LFA 上进行肉眼检测。变异体通过 Sanger 测序得到确认,LFA 检测方法的灵敏度和特异性通过 Agena MassARRAY® 技术得到验证:结果:在103名高胆固醇血症患者中,5人(4.8%)的E101K和LDLR突变检测结果呈阳性,其余人(包括健康对照组)的检测结果呈阴性。这一结果与桑格测序和 Agena MassARRAY® 的结果一致。由于 DNA 诊断得到证实,这五个人可被归类为 "确定的 FH"。与使用 Agena MassARRAY® 进行基因分型的结果相比,用 LFA 检测变异的灵敏度和特异性均为 100%:结论:开发的 LFA 可用于 POC 环境下检测 LDLR 基因中的 E101K 变异。该 LFA 还可用于筛查 LDLR 基因中 E101K 变异的家庭成员,并适用于其他 SNP 的检测。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Development of DNA-Based Lateral Flow Assay for Detection of LDLR Gene Mutation for Familial Hypercholesterolemia.

Background: The techniques for detecting single nucleotide polymorphisms (SNP) require lengthy and complex experimental procedures and expensive instruments that may only be available in some laboratories. Thus, a deoxyribonucleic acid (DNA)-based lateral flow assay (LFA) was developed as a point-of-care test (POCT) diagnostic tool for genotyping. In this study, single nucleotide variation (E101K) in the low-density lipoprotein receptor (LDLR) gene leading to familial hypercholesterolemia (FH) was chosen as a model.

Methods: Hypercholesterolemic individuals (n = 103) were selected from the Malaysian Cohort project (UKM Medical Molecular Biology Institute) while the control samples were selected from the Biobank (UKM Medical Molecular Biology Institute). The DNA samples were isolated from whole blood. Polymerase chain reaction (PCR) amplification process was performed using bifunctional labelled primers specifically designed to correspond to the variant that differentiates wild-type and mutant DNA for visual detection on LFA. The variant was confirmed using Sanger sequencing, and the sensitivity and specificity of the LFA detection method were validated using the Agena MassARRAY® technique.

Results: Out of 103 hypercholesterolemic individuals, 5 individuals (4.8%) tested positive for E101K, LDLR mutation and the rest, including healthy control individuals, tested negative. This result was concordant with Sanger sequencing and Agena MassARRAY®. These five individuals could be classified as Definite FH, as the DNA diagnosis was confirmed. The sensitivity and specificity of the variant detection by LFA is 100% compared to results using the genotyping method using Agena MassARRAY®.

Conclusion: The developed LFA can potentially be used in the POC setting for detecting the E101K variant in the LDLR gene. This LFA can also be used to screen family members with E101K variant in the LDLR gene and is applicable for other SNP's detection.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
Malaysian Journal of Medical Sciences
Malaysian Journal of Medical Sciences MEDICINE, RESEARCH & EXPERIMENTAL-
CiteScore
2.70
自引率
0.00%
发文量
89
审稿时长
9 weeks
期刊介绍: The Malaysian Journal of Medical Sciences (MJMS) is a peer-reviewed, open-access, fully online journal that is published at least six times a year. The journal’s scope encompasses all aspects of medical sciences including biomedical, allied health, clinical and social sciences. We accept high quality papers from basic to translational research especially from low & middle income countries, as classified by the United Nations & World Bank (https://datahelpdesk.worldbank.org/knowledgebase/ articles/906519), with the aim that published research will benefit back the bottom billion population from these countries. Manuscripts submitted from developed or high income countries to MJMS must contain data and information that will benefit the socio-health and bio-medical sciences of these low and middle income countries. The MJMS editorial board consists of internationally regarded clinicians and scientists from low and middle income countries.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信