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.
出处
《分析科学学报》
CAS
CSCD
北大核心
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)
