为了对掺入猪肉成分的比例进行鉴定，本研究拟建立基于同源加尾系统（Homo-tag Assisted Non-dimer system，HANDS）的双重实时PCR检测方法，借助简易数学模型将实时PCR的Ct值（循环阈值）换算成猪肉成分的百分比。根据常见家畜、家禽染色体中的管家基因Myostatin序列设计家禽家畜通用引物和探针序列，针对猪染色体beta actin基因设计猪源性成分特异型引物和探针序列，并在所有引物5’端加上同源引物。通过评估以上两套引物和探针的通用性，特异性，优化引物和探针浓度、退火温度，构建同源加尾双重实时PCR检测方法对不同猪源性成分比例的肉样进行检测并换算Ct值得出混合肉样中猪源性成分的比例。所构建的同源加尾双重实时PCR方法两重之间扩增效率相近，通用性引物、探针可以扩增所有家畜家禽成分，特异性引物探针只扩增猪源性成分。通过检测制备的混合肉样显示，该方法换算结果与肉样实际比例接近。

The beef & mutton and their products in the market always mixed with pork, which is relatively cheaper. In order to identify the proportion of pork, a dual real-time PCR assay was established on the basis of Homo-tag Assisted Non-dimer system (HANDS). The assay applied a simple mathematical maodel to calculate the percentage of pork fraction according to the Ct (cycle thresholds) values shown on the real-time PCR assay. Universal primers and probe sequences were designed according to the House-keeping gene that consist in all livestock-poultry chromosome named Myostatin, while specific primers and probe sequences were designed according to the beta actin gene sequences in swine chromosome. The homologous tag primer sequences were added to the 5’-end of all primer sequences. To establish the real-time PCR assay, the universality and specificity of the primers & probes were assessed, and the concentration of primers & probe and annealing temperature of reaction were optimized. After testing of 5 mixed meats which were prepared in advance, the calculation of pork proportion was based on the Ct values which were obtained from the assay. Comparing the proportion differences between the calculation and actual preparation, the feasibility of identification pork proportion in mixed meats was evaluated. The efficiency of amplification of two reactions in dual Real-Time PCR detection is approximate. The calculation results are in agreement with the actual proportion according to the detection results of the mixed meats which have been prepared in advance. The newly established assay is stable, sensitive and accurate, so it is suitable for the supervision of food in market.

原国家质量监督检验检疫总局科技项目（2016IK299）