牛奶β-乳球蛋白质实时荧光定量PCR检测方法的建立  

Establishment of a Taqman Real-Time Quantitative PCR for the Detection of β-Lactoglobulin Gene

作　　者：贾敏[1] 张银志[2] 张亦凡[3] 孙秀兰[1,2] 

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

出　　处：《食品与生物技术学报》2015年第6期605-612,共8页Journal of Food Science and Biotechnology

基　　金：国家"十二五"科技支撑计划项目(2011BAK10B03)

摘　　要：建立一种快速、特异、灵敏的Taqman实时荧光定量PCR（real-time PCR）方法,用于牛奶主要过敏原β-乳球蛋白质的检测。根据Gen Bank登录的牛β-乳球蛋白质的DNA序列设计,合成一对特异性引物和探针。将扩增产物连接到p MD19-T载体上,制备质粒标准品并测序鉴定,10倍梯度稀释含有β-乳球蛋白质基因的重组质粒,进行实时荧光定量PCR扩增,绘制标准曲线,检测该方法的特异性、稳定性、灵敏性,同时将建立的方法用于10种市售食品的检测。成功建立了β-乳球蛋白质的实时荧光定量PCR检测方法,标准曲线的Ct值与模板浓度在3.18×103~3.18×107copies范围内线性关系良好,R2值为0.997 8;检测灵敏度高（318 copies/μL）;特异性强,对羊奶、豆浆DNA均无扩增反应;稳定性好,组内、组间的变异系数均在5%以内。对10种食品牛奶过敏原的检测结果与标签相符。表明所建立的实时荧光定量PCR方法可应用于食品中牛奶过敏原β-乳球蛋白质的检测并可推广到其它过敏原的检测。The research aimed to establish a Taqman real-time fluorescence quantitative assay for the rapid detection of milk allergenβ-lactoglobulin in food. Specific primers and Taqman probe were first designed based on the gene sequence of β-lactoglobulin for real-time PCR. The purified DNA product was linked with the pMDl9-T vector to construct recombined plasmids as a standard PCR template. Plasmids were then identified with colony PCR and subjected to sequencing. Real-time fluorescence PCR assay was performed with plasmids and the standard curve was constructed with DNA copies and Ct. Sensitivity, specificity, reproducibility and application of the real-time method were also evaluated. Results showed that the β-lactoglobulin DNA fragment was successfully cloned and the standard curve had a good linear relationship ranging from 3.18 × 103 to 3.18 × 107 copies. The detection sensitivity reached up to 318 copies/μL. Furthermore, specificity and stability of the method were good. Ten foods were detected with the β-Lg DNA residue and the results were consistent well with their allergen labels. Therefore, the method may become a complementary tool for milk allergen detection and it is applicable for other food allergens.

关 键 词：牛奶过敏原质 β-乳球蛋白质 Taqman实时荧光定量PCR法 检测 

分 类 号：TS252.7[轻工技术与工程—农产品加工及贮藏工程]