The invention belongs to the technical field of biomedicine. Abnormal methylation of genes involved in cancer development, the aim of the invention is to overcome the methylation sensitive restriction endonuclease method, bisulfite sequencing and methylation specific PCR method shortcomings, provides a simple operation and high sensitivity of HPP1 gene methylation quantitative detection method. The present invention from pancreatic cancer, extracted from the standard curve samples prepared by DNA ACTB, using the full methylated template, the standard curve of sample preparation of HPP1; extraction of tissue to be measured DNA, and chemical modification, human HPP1 gene promoter CPG island methylation primer and probe design, the needle of ginseng ACTB gene CPG Island in the promoter region of BSP primer design and probe, using real-time quantitative PCR method of Taqman MGB real-time PCR of sulfite treated DNA, calculate the quantitative value of HPP1 gene methylation. The method of the invention is fast, accurate and sensitive.
【技术实现步骤摘要】
【技术保护点】
一种ＨＰＰ１基因甲基化定量检测方法，其特征在于该方法包括如下步骤： Ａ、待检样本处理：用ＱＩＡＧＥＮ ＥｐｉＴｅｃｔ Ｂｉｓｕｆｉｔｅ ＫｉｔＴＭ试剂盒对待检样本的ＤＮＡ进行重亚硫酸盐处理，用作扩增模板； Ｂ、分别制备ＡＣＴＢ参照基因的标准品和ＨＰＰ１靶基因的标准品： 设计并合成ＡＣＴＢ参照基因的ＢＳＰ引物： ＡＣＴＢ ＢＦ：５′ＴＧＧＴＧＡＴＧＧＡＧＧＡＧＧＴＴＴＡＧＴＡＡＧＴ ３′ ＡＣＴＢ ＢＲ：５′ＡＡＣＣＡＡＴＡＡＡＡＣＣＴＡＣＴＣＣＴＣＣＣＴＴＡＡ ３′设计并合成ＨＰＰ１靶基因的ＭＳＰ引物： ＨＰＰ １ＢＦ：５′ＡＧＴＡＧＴＡＧＴＡＧＧＧＴＡＧＡＧＡＧＧＧＧ′３′ ＨＰＰ １ＢＲ：５′ＡＡＣＣＴＴＡＡＡＡＴＴＡＣＡＣＲＣＴＣＴＴ ３′ ＡＣＴＢ参照基因进行ＢＳＰ反应的条件：９５℃预变性１０ｍｉｎ；９５℃变性３０ｓ．６０℃退火３０ｓ．７２℃延伸３０ｓ，共４０个循环； ＨＰＰ１靶基因进行ＢＳＰ反应的条件：９５℃预变性１０ｍｉｎ；９５℃变性３０ｓ．５３．３℃退火３０ｓ．７２℃延伸３０ｓ，共４０个循环； ＰＣＲ产物进行纯化，连接入ｐＭＤ１８－Ｔ Ｖｅｃｔｏｒ，然...
【技术特征摘要】
【专利技术属性】
技术研发人员：李兆申，王小玮，高军，杜奕奇，龚燕芳，
申请(专利权)人：中国人民解放军第二军医大学，
类型：发明
国别省市：31[中国|上海]
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