一种用于实时荧光PCR法检测肉制品中鼠源性成分特异性引物的研发

Development of specific primers for determination of mouse-derived ingredients in meat products by real-time fluorescent PCR

目的研发一种用于实时荧光PCR法检测肉制品中鼠源性成分的特异性引物。方法通过比对NCBI中多种物种的脱氧核糖核酸细胞色素b(mitochondrial deoxyribonucleic acid cytochrome b,Cytb)基因全序列,设计针对鼠源性成分的特异性引物。采用DNA提取试剂盒提取鼠DNA,以其为模板,通过设计的特异性引物进行实时荧光PCR扩增,试验重复3次。同时验证该引物的特异性,并对羊肉卷、牛肉卷和猪肉片样品进行检测。结果通过设计的特异性引物进行3次实时荧光PCR扩增的Cp平均值为16.47,标准差为0.430,熔解曲线具有明显的单一主峰,且重合度良好。仅鼠源DNA模板的PCR产物可见115 bp的目的条带,其他物种DNA模板均未扩增出该目的条带。羊肉卷、牛肉卷和猪肉片样品中均未检测出鼠源性成分。结论本实验设计的引物可用于实时荧光PCR法对肉制品中鼠源性成分的检测,且特异性较好。

Objective To develop a pair of specific primers for determination of mouse-derived ingredients in meat products by real-time fluorescent PCR. Methods Specific primers were designed according to the whole sequence of mitochondrial deoxyribonucleic acid cytochrome b（Cytb）gene in NCBI and used for amplification by real-time fluorescent PCR using the mouse DNA extracted by DNA extraction kit as a template. The test was repeated for 3 times,while the primers were verified for specificity and used for determination of mouse-derived ingredients in lamb kebab roll,beef roll and pork slices. Results The mean Cp value of three cycles of real-time fluorescent PCR with the designed primers was16. 47,with a standard deviation of 0. 430. A single main peak was observed on melting curve,with a high coincidence degree. The target band at a length of 115 bp was amplified only from the mouse-derived DNA template but not from the DNA templates for other animal origins. No mouse-derived ingredients were detected in lamb kebab roll,beef roll or pork slices. Conclusion The designed primers may be used for the determination of mouse-derived ingredients in meat products by real-time fluorescent PCR,which showed high specificity.