基于扩增阻滞突变系统-聚合酶链式反应法的A1/A2牛奶鉴别试剂盒的开发及应用

Development and application of A1/A2 milk identification kits based on amplification refractory mutation system polymerase chain reaction

中文关键词:  A2牛奶  β-酪蛋白  扩增阻滞突变系统PCR技术  鉴别  试剂盒

英文关键词:A2 milk  β-casin  amplification refractory mutation system polymerase chain reaction  authentication  kit

中文摘要:

      目的 基于扩增阻滞突变系统-聚合酶链式反应(amplification refractory mutation system polymerase chain reaction, ARMS-PCR)技术, 开发A1A2牛奶鉴别试剂盒, 并对市场上A2牛奶产品进行检测应用, 实现对A2牛奶的鉴别检测。方法 本研究先利用D-loop基因片段设计了牛内参基因引物探针组(标记FAM荧光)。随后, 在β-酪蛋白基因突变位点分别设计了A1基因引物探针组和A2基因引物探针组(均标记VIC荧光)。基于ARMS-PCR技术构建了双通道ARMS-PCR反应体系与程序, 进而成功开发了A1A2牛奶实时荧光ARMS-PCR试剂盒。为进一步验证试剂盒性能, 将其应用于市场上A2牛奶的检测, 并将检测结果与DNA测序法结果对比。结果 本试剂盒特异性强, 检测DNA灵敏度为0.1 ng/L; 10份市售实际样品的应用检测结果与测序结果一致。结论 本试剂盒特异性好, 灵敏度高、准度度高, 适用于A2牛奶的真实性鉴别。

英文摘要:

      Objective To achieve accurate identification and detection of A2 milk, an A1A2 milk identification kit based on the amplification refractory mutation system polymerase chain reaction (ARMS-PCR) was developed and applied to the testing of A2 milk products in the market. Methods The study first utilized the D-loop gene to design primers and probes for bovine internal reference genes labeled with FAM fluorescence. Subsequently, A1 gene primer probe group and A2 gene primer probe group were designed for the mutation sites of the β-casein gene, both labeled with VIC fluorescence. Accordingly, a dual-channel ARMS-PCR reaction system and procedure based on ARMS-PCR technology was constructed, leading to the successful development of the A1A2 milk real-time fluorescence ARMS-PCR kit. To further validate the performance of the kit, it was applied to the testing of commercially available A2 milk samples, and the results obtained were compared with those obtained from DNA sequencing. Results This kit demonstrated high specificity with a detection sensitivity of 0.1 ng/L, and the application testing results on 10 commercially available samples were consistent with DNA sequencing results. Conclusion The kit shows excellent specificity, high sensitivity and accuracy, making it suitable for authenticating A2 milk products.