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

DOI：10.3969/j.issn.1673-1689.2015.06.007

中文关键词: 牛奶过敏原质 β-乳球蛋白质 Taqman 实时荧光定量PCR法 检测

英文关键词: milk allergen, β-lactoglobulin, Taqman real-time quantitative PCR, detection

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中文摘要:

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GenBank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pMD19-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。

英文摘要:

The research aimed to establish a Taqman real-time fluorescence quantitative assay for the rapid detection of milk allergen β-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.

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