Establishment And Application Of PCR Assay For Animal-derived Foods

In recent years,food safety is a more sensitive topic.The problem of food safety is particularly acute,which is dominated by animal-derived ingredients.The adulteration of meat products has become the focus of consumers' attention.Due to the diversity of adulteration methods,it is difficult to detect the source of meat components by relying solely on traditional methods of identification.(1)In this study,pigs,cattle,sheep,chickens,and ducks were used to design Cytb DNA-specific primers for cloning and sequencing.The lengths of the amplified product fragments were 211 bp,100 bp,160 bp,126 bp,and 110 bp.The amplified product fragments compared with NCBI GenBank Blast analysis,homology is 100%,indicating that the five amplified fragments are the target sequence.The specificity and sensitivity of the 5 primers were confirmed by conventional PCR.The sensitivity was 2.0 ng/?L of bovine template DNA,i.e.1.0%,and 0.2 ng/?L for pig,sheep,chicken and duck template DNA,i.e.0.1%.The five primers can be used as real-time fluorescent PCR primers.Routine PCR was used to detect 9 kinds of meat products on the market.The results showed that there were pig-derived or duck-derived components in mutton rolls and beef rolls respectively.Chicken-derived or duck-derived ingredients were found in different types of pork sausages.(2)Using the SYBR Green II qRT-PCR method,the establishment of a standard curve and the detection of the specificity and sensitivity of the primers showed that the specificity and sensitivity of the five primers met the test requirements.The sensitivities were 0.2 ng/?L for bovine template DNA,i.e.0.1%,and 0.02 ng/?L for pigs,sheep,chickens and ducks,i.e.0.01%.The test results for 9 meat products were consistent with conventional PCR and further detection of doped meat components not detected by conventional PCR.The two detection systems established in this study,the real-time fluorescence PCR method simplifies the conventional PCR method,can detect the lower template DNA concentration.The detection results are more accurate,can be completed in about 1 h.The method can be easy promotion in inspection practice.