阿尔茨海默病患者载脂蛋白E 3种分型方法的比较

Comparative study of three genotyping methods for apolipoprotein E in Alzheimer′s disease

目的采用竞争性等位基因特异性PCR(KASP)法、单碱基末端延伸(SNaPshot)法对载脂蛋白E(APOE)进行分型检测,并与Sanger测序法比较,以期获得更为高效、稳定、经济的中、高通量APOE分型手段。方法选取既往收集的覆盖全部6种常见APOE分型的阿尔茨海默病(AD)及轻度认知障碍(MCI)患者全血核酸样本48份,根据KASP法和SNaPshot法技术原理设计实验识别APOE 2个关键单核苷酸多态性(SNP)位点rs429358和rs7412,并对样本进行APOE分型检测。调整样本顺序重复检测以验证方法的稳定性和可重复性。扩大样本量,收集AD、MCI患者全血样本107份,采用上述两种方法进行APOE分型检测,Sanger测序法验证。结果采用KASP法和SNaPshot法对48份已知APOE分型样本的2次检测结果与原分型完全一致。进一步扩大样本量对107例样本进行分型检测,与Sanger测序法得到的结果完全一致。结论采用KASP法和SNaPshot法进行APOE分型检测具有快速、准确、结果直观等特点,应用中、高通量APOE分型检测相对于Sanger测序法效率更高、成本更低,具有一定推广应用价值。

Objective To develop a more efficient,stable and economical medium and high throughout method for detecting apolipoprotein E(APOE)genotypes based on kompetitive allele specific PCR(KASP)and SNaPshot methods and with the comparison with Sanger sequencing.Methods In this study,a total of 48 whole blood nucleic acid sample of Alzheimer′s disease(AD)patients or mild cognitive impairment(MCI)patients with 6 kinds of APOE genotypes were selected.According to the principle of KASP and SNaPshot technology,experiments were designed to identify two key single nucleotide polymorphism(SNP)sites rs429358 and rs7412 of APOE,and APOE genotypes was carried out on the samples.The sample sequence was adjusted and the test was repeated to verify the stability and repeatability of the method.The sample size was expanded,another 107 whole blood samples from patients with AD and MCI were collected,and the above two methods for APOE genotypes were used.The results were validated by Sanger sequencing.Results There was complete concordance in the APOE genotypes between the two detection results by KASP and SNaPshot methods in 48 samples.Further on 107 samples,the results were completely consistent with those obtained by Sanger sequencing method.Conclusion The KASP and SNaPshot methods for APOE genotypes have the advantages of rapid,accurate,intuitive and so on.Compared to Sanger sequencing,the new methods are more efficient and cost-effective especially for medium or high throughput APOE genotypes,which have certain popularization and application value.