牛奶α-乳白蛋白基因实时荧光定量PCR检测方法的建立  

Establishment of A Real-time Quantitative PCR Method for Detection of α-Lactalbumin Gene

作　　者：贾敏[1] 张亦凡[2] 张银志[3] 孙秀兰[1,3] 

机构地区：[1]江南大学食品学院,江苏无锡214122 [2]陕西省汉中市产品质量监督检验所,陕西汉中723000 [3]江南大学食品科学与技术国家重点实验室,江苏无锡214122

出　　处：《分析科学学报》2015年第2期165-170,共6页Journal of Analytical Science

基　　金："十二五"国家科技支撑计划(No.2011BAK10B03)

摘　　要：采用Taqman探针技术,建立食品中牛奶过敏原α-乳白蛋白基因的实时荧光定量PCR检测方法。根据α-乳白蛋白基因序列设计特异性引物及Taqman探针进行PCR扩增,构建质粒,经酶切鉴定测序后,建立拷贝数（copies）-Ct标准曲线。成功克隆α-乳白蛋白目的基因,建立的标准曲线在1.12×10^3-1.12×10^8 copies范围内线性关系良好,灵敏度高,液体样品检测限达到1 000copies/mL,特异性强,稳定性好。该法可用于实际商品中牛奶过敏原α-乳白蛋白组分的定量检测。A Taqman real-time fluorescence quantitative assay for the rapid detection of milk allergenα-lactalbumin in food was established.PCR primers and Taqman probe were designed based on the gene sequence ofα-lactalbumin（GenBank No.AF249896.1）for real-time PCR.Plasmids were prepared as the standard PCR template and identified with enzyme digestion and then were subjected to sequencing.Then real-time fluorescence PCR assay was performed.Theα-lactalbumin DNA fragment has been cloned successfully and the standard curve had a good linear relationship ranging from 1.12×103 to 1.12×108copies.The detection sensitively of liquid sample reached up to 1 000copies/mL.Furthermore,the specificity and stability of the method were good.The real-time PCR method has been developed successfully and it could be applied toα-lactalbumin detection with high sensitivity and specificity.

关 键 词：牛奶 Α-乳白蛋白 Taqman实时荧光定量PCR 

分 类 号：O657.3[理学—分析化学]