酶联免疫法检测MTHFRC677T位点基因多态性

目的建立一种基于错配杂交及酶联免疫检测亚甲基四氢叶酸还原酶基因多态性的新方法。方法针对多态位点设计两条寡核苷酸探针,分别与野生型及突变型序列互补。将两条地高辛标记的探针分别与含多态位点C677T的PCR扩增产物杂交,然后显色检测,根据两条探针的吸光度值之比确定样品的基因型。结果用本法随机检测了50例DNA样品,结果三种基因型的发光值之比可以严格区分,其中野生型(C/C)15例,杂合型(C/T)28例,突变型(T/T)7例,与参考方法(PCR-RFLP)的检测结果完全一致。结论本方法操作简便、检测快速而且费用低廉,可广泛应用于MTHFR基因突变及多态性检测。

Detection of Methylenetetrahydrofolate Reductase Polymorphisms with the Colorimetric Method Based on Mismatch Hybridization

Objective: To establish a new method for rapid detection polymorphisms of methylenetetrahydrofolate reductase with a colorimetric method based on mismatch hybridization.Methods: Two oligonucleotides probes for MTHFR polymorphic site were designed,which perfectly match the wild-type and mutant genotype of C677T site,respectively.After hybridization with amplified DNA fragment,the hybrids were detected with a colorimetric method and the genotype were identified by calculating the ratio of absorbent obtained with two probes.Results: The polymorphism of 50 samples were analysed by the method.Wild-type,heterozygous and homozygous were detected in 15 cases,28 cases and 7 cases,respectively.Genotypes of the 50 samples is accordance with the results detected by PCR-RFLP.Conclusion: This method is simple and rapid,and can be widely used for analysis gene mutation.