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. 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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.
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.
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