Saidi Lina Khalida, Md Rani Zam Zureena, Sulaiman Siti Aishah, Jamal Rahman, Ismail Aziah, Alim Anis Amirah, Ayob Sharipah Nadzirah Syed Ahmad, Dee Chang Fu, Hamzah Azrul Azlan, Abdul Murad Nor Azian
UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.
Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Kelantan, Malaysia.
Malays J Med Sci. 2024 Jun;31(3):92-106. doi: 10.21315/mjms2024.31.3.6. Epub 2024 Jun 27.
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 gene leading to familial hypercholesterolemia (FH) was chosen as a model.
Hypercholesterolemic individuals ( = 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.
Out of 103 hypercholesterolemic individuals, 5 individuals (4.8%) tested positive for E101K, 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.
The developed LFA can potentially be used in the POC setting for detecting the E101K variant in the gene. This LFA can also be used to screen family members with E101K variant in the gene and is applicable for other SNP's detection.
检测单核苷酸多态性（SNP）的技术需要冗长复杂的实验程序以及昂贵的仪器，而这些仪器可能仅在某些实验室才有。因此，开发了一种基于脱氧核糖核酸（DNA）的侧向流动分析（LFA）作为用于基因分型的即时检验（POCT）诊断工具。在本研究中，选择导致家族性高胆固醇血症（FH）的低密度脂蛋白受体基因中的单核苷酸变异（E101K）作为模型。
从马来西亚队列项目（UKM医学分子生物学研究所）中选取高胆固醇血症个体（n = 103），而对照样本则从生物样本库（UKM医学分子生物学研究所）中选取。从全血中分离DNA样本。使用专门设计的双功能标记引物进行聚合酶链反应（PCR）扩增过程，该引物对应于区分野生型和突变型DNA的变体，以便在LFA上进行视觉检测。使用桑格测序法确认变体，并使用Agena MassARRAY技术验证LFA检测方法的灵敏度和特异性。
在103名高胆固醇血症个体中，有5名个体（4.8%）的E101K突变检测呈阳性，其余个体，包括健康对照个体，检测呈阴性。该结果与桑格测序和Agena MassARRAY结果一致。这五名个体可被归类为确诊的FH，因为DNA诊断得到了证实。与使用Agena MassARRAY的基因分型方法的结果相比，LFA检测变体的灵敏度和特异性为100%。
所开发的LFA有可能用于即时检验环境中检测该基因中的E101K变体。这种LFA还可用于筛查该基因中具有E101K变体的家庭成员，并且适用于其他SNP的检测。
