ApoE Genotyping By Direct Amplification Method With Whole Blood And Its Clinical Applications

Apolipoprotein E(ApoE)is a protein which combines with lipids in the body to form molecules called lipoproteins.Lipoproteins are responsible for packaging cholesterol and other fats and carrying them through the bloodstream.It has been reported that the polymorphism of the ApoE gene is related to the metabolism of Alzheimer’s disease,of atherosclerosis and of lipid-lowering drugs.Currently,genotyping methods for ApoE such as sequencing,the polymerase chain reaction-restriction fragment length polymorphism assay,the polymerase chain reaction-single strand conformation polymorphism assay,etc.,already used in biological and medical laboratories,but these methods are costly and time-consuming,the operational procedures are complex and they require large instruments.This limits the clinical application of these methods.Based on the ApoE gene,this study combines the amplification refractory mutation system with the lateral flow assay system and establishes a genotyping method.This method can be directly used for PCR amplification only by simple pretreatment of whole blood.We also developed a device for reading the results.After amplification,the results can be read in just 5 minutes using the reader.Our method does not require DNA extraction,and it takes 90 minutes to complete the entire experiment.It has a sensitivity as low as 1 ng genomic DNA.Fifty clinical samples were tested with our method and the results were compared with sequencing.The coincidence rate of the two methods was 100%,indicating that the method has a high accuracy.Thirty-five cases of special clinical samples(with high bilirubin,high cholesterol,high triglycerides,or hemolytic samples,samples with low numbers or high numbers of white blood cells,blood disease patients),the detection rate remained 100%,indicating that this method is not affected by these potentially interfering factors.After applying the method to 327 ischemic stroke and 256 control samples there appeared to be no correlation between the ApoE gene polymorphisms and the risk of ischemic stroke.The method is accurate,sensitive,low cost and simple in operation.ApoE genotyping with our method can be used to provide a basis for early prevention of Alzheimer’s disease in multi-level medical institutions.It can also be used for clinical research of other diseases related to the ApoE genotypes.