Study on the Identification of Pork Fraction Proportion in Mixed Meats with Dual Real-time PCR Based on Homo-tag Assisted Non-dimer System
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Abstract:
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.
