CAJ | 학술논문

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0.9978；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。
건립일충쾌속、특이、령민적Taqman실시형광정량PCR（real-time PCR）방법，용우우내주요과민원β-유구단백질적검측。근거GenBank등록적우β-유구단백질적DNA서렬설계，합성일대특이성인물화탐침。장확증산물련접도pMD19-T재체상，제비질립표준품병측서감정，10배제도희석함유β-유구단백질기인적중조질립，진행실시형광정량PCR확증，회제표준곡선，검측해방법적특이성、은정성、령민성，동시장건립적방법용우10충시수식품적검측。성공건립료β-유구단백질적실시형광정량PCR검측방법，표준곡선적Ct치여모판농도재3.18×103～3.18×107copies범위내선성관계량호，R2치위0.9978；검측령민도고(318 copies/μL)；특이성강，대양내、두장DNA균무확증반응；은정성호，조내、조간적변이계수균재5%이내。대10충식품우내과민원적검측결과여표첨상부。표명소건립적실시형광정량PCR방법가응용우식품중우내과민원β-유구단백질적검측병가추엄도기타과민원적검측。
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.