PDF(Pubmed)
Abstract：
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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®.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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®.
UNASSIGNED: 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.
摘要：
■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，这些仪器可能仅在某些实验室中可用。因此，我们开发了一种基于脱氧核糖核酸(DNA)的侧流检测(LFA)作为基因分型的即时检测(POCT)诊断工具.在这项研究中,选择导致家族性高胆固醇血症（FH）的低密度脂蛋白受体（LDLR）基因的单核苷酸变异（E101K）作为模型。
■高胆固醇血症个体(n=103)选自马来西亚队列项目(UKM医学分子生物学研究所),而对照样品选自生物库(UKM医学分子生物学研究所)。从全血中分离DNA样品。使用双功能标记的引物进行聚合酶链反应（PCR）扩增过程，该引物专门设计为对应于区分野生型和突变体DNA的变体，以在LFA上进行视觉检测。使用Sanger测序证实了该变体，使用AgenaMassARRAY®技术验证了LFA检测方法的敏感性和特异性。
■在103名高胆固醇血症个体中，5人（4.8%）E101K检测呈阳性，LDLR突变和其余的，包括健康的控制者，测试为阴性。该结果与Sanger测序和AgenaMassARRAY®一致。这五个人可以被归类为确定的FH，DNA诊断得到证实。与使用AgenaMassARRAY®的基因分型方法的结果相比,通过LFA的变体检测的灵敏度和特异性为100%。
■开发的LFA可以潜在地用于POC设置中,用于检测LDLR基因中的E101K变体。该LFA还可用于筛选LDLR基因中具有E101K变体的家族成员,并且适用于其他SNP的检测。
■高胆固醇血症个体(n=103)选自马来西亚队列项目(UKM医学分子生物学研究所),而对照样品选自生物库(UKM医学分子生物学研究所)。从全血中分离DNA样品。使用双功能标记的引物进行聚合酶链反应（PCR）扩增过程，该引物专门设计为对应于区分野生型和突变体DNA的变体，以在LFA上进行视觉检测。使用Sanger测序证实了该变体，使用AgenaMassARRAY®技术验证了LFA检测方法的敏感性和特异性。
■在103名高胆固醇血症个体中，5人（4.8%）E101K检测呈阳性，LDLR突变和其余的，包括健康的控制者，测试为阴性。该结果与Sanger测序和AgenaMassARRAY®一致。这五个人可以被归类为确定的FH，DNA诊断得到证实。与使用AgenaMassARRAY®的基因分型方法的结果相比,通过LFA的变体检测的灵敏度和特异性为100%。
■开发的LFA可以潜在地用于POC设置中,用于检测LDLR基因中的E101K变体。该LFA还可用于筛选LDLR基因中具有E101K变体的家族成员,并且适用于其他SNP的检测。
