Human apolipoprotein E (ApoE) is associated with high cholesterol levels, coronary artery disease, and especially Alzheimer’s disease. In this study, we developed an ApoE genotyping and one-step multiplex polymerase chain reaction (PCR) based-capillary electrophoresis (CE) method for the enhanced diagnosis of Alzheimer’s. The primer mixture of ApoE genes enabled the performance of direct one-step multiplex PCR from whole blood without DNA purification. The combination of direct ApoE genotyping and one-step multiplex PCR minimized the risk of DNA loss or contamination due to the process of DNA purification. All amplified PCR products with different DNA lengths (112-, 253-, 308-, 444-, and 514-bp DNA) of the ApoE genes were analyzed within 2 min by an extended voltage programming (VP)-based CE under the optimal conditions. The extended VP-based CE method was at least 120–180 times faster than conventional slab gel electrophoresis methods In particular, all amplified DNA fragments were detected in less than 10 PCR cycles using a laser-induced fluorescence detector. The detection limits of the ApoE genes were 6.4–62.0 pM, which were approximately 100–100,000 times more sensitive than previous Alzheimer’s diagnosis methods In addition, the combined one-step multiplex PCR and extended VP-based CE method was also successfully applied to the analysis of ApoE genotypes in Alzheimer’s patients and normal samples and confirmed the distribution probability of allele frequencies. This combination of direct one-step multiplex PCR and an extended VP-based CE method should increase the diagnostic reliability of Alzheimer’s with high sensitivity and short analysis time even with direct use of whole blood.

人载脂蛋白E（ApoE）与高胆固醇水平，冠状动脉疾病，尤其是阿尔茨海默氏病有关。在这项研究中，我们开发了基于ApoE的基因分型和基于一步法的多重聚合酶链反应（PCR）的毛细管电泳（CE）方法，以增强对阿尔茨海默氏病的诊断。ApoE基因的引物混合物无需DNA纯化即可从全血进行直接一步多重PCR。直接ApoE基因分型和一步多重PCR的结合将由于DNA纯化过程而导致的DNA丢失或污染的风险降到最低。在最佳条件下，通过基于扩展电压编程（VP）的CE在2分钟内分析了所有具有不同DNA长度（112-，253-，308-，444-和514-bp DNA）的扩增的PCR产物。条件。扩展的基于VP的CE方法比传统的平板凝胶电泳方法快至少120–180倍。特别是，使用激光诱导的荧光检测器在不到10个PCR循环中就检测到了所有扩增的DNA片段。ApoE基因的检出限为6.4–62.0 pM，比以前的阿尔茨海默氏病诊断方法灵敏度高约100–100,000倍。此外，单步多重PCR和基于VP的扩展CE方法相结合也已成功应用于阿尔茨海默氏病患者和正常样本中ApoE基因型的分析，并证实了等位基因频率的分布可能性。