基于Cycling探针的实时荧光聚合酶链式反应检测河鲀鱼中掺杂的横纹东方鲀成分

Detection of the components of Takifugu oblongus in puffer fish by real-time fluorescent polymerase chain reaction based on Cycling probe

中文关键词:  关键词：河鲀鱼  食品真实性；横纹东方鲀；Cycling探针；实时荧光聚合酶链式反应

英文关键词:puffer fish  food authenticity  Takifugu oblongus  Cycling probe  real-time fluorescent polymerase chain reaction

中文摘要:

      目的 开发基于Cycling探针实时荧光聚合酶链式反应(real-time fluorescent polymerase chain reaction, RT-PCR)用于检测河鲀鱼中横纹东方鲀(Takifugu oblongus, T. oblongus)成分。方法 基于细胞色素C氧化酶亚型I (cytochrome C oxidase subunit I, COI)基因为靶标设计RT-PCR引物及Cycling探针, 开发横纹东方鲀成分检测方法, 评价方法的特异性、扩增效率、灵敏度和稳定性。结果 横纹东方鲀成分与密切相关的其他8种河鲀鱼、4种其他鱼类物种DNA无交叉反应, 具有很好的特异性; 方法扩增效率为93.47%; 方法重复18次均给出阳性报告的最小稀释点(limit of detection 6, LOD6)为14.64 pg/μL(相对应的质量含量为0.1%), 95%置信水平条件下检出限为34.41 pg/μL (11.59~119.05 pg/μL, LOD95%), 稳定性分析(P=0.152)表明该方法可转移到其他实验室以及在常规分析中使用。结论 本研究开发的Cycling探针RT-PCR方法可对河鲀鱼食品中掺杂0.1%的横纹东方鲀成分进行可靠追溯。

英文摘要:

      Objective To develop a real-time fluorescent polymerase chain reaction (RT-PCR) based on Cycling probe to detect the components of Takifugu oblongus (T. oblongus). Methods RT-PCR primer and Cycling probe were designed based on cytochrome C oxidase subunit I (COI) gene as the target, and the method was developed to detect the components of T. oblongus. The specificity, amplification efficiency, sensitivity and stability of the method were evaluated. Results Specificity was tested using genomic DNA from 8 kinds of species puffer fish (Lagocephalus and Takifugu) that closely associated with the target (T. oblongus) and 4 kinds of other species fish. There was no cross reaction, it verified the accuracy and specificity of the method; the amplification efficiency of the method was 93.47%. The limit of detection 6 (LOD6) of the method was 14.64 pg/μL (the corresponding mass content was 0.1%), the detection limit at 95% confidence level was 34.41 pg/μL (11.59-119.05 pg/μL, LOD95%). Stability analysis (P=0.152) showed that the method could be transferred to other laboratories and used in routine analysis. Conclusion The Cycling probe RT-PCR method developed in this study can reliably trace 0.1% of T. oblongues in puffer fish.