Study On Determination Of Pig-derived Components In Food By Two PCR

With the development of the social economy,while the dietary structure of people has changed dramatically,the food industry is also facing more and more challenges.Incidents of food-related violations such as adulteration of animal-derived ingredients have had a significant impact on consumer health and the market economy.In order to better detect adulteration of animal-derived ingredients in food,protect consumer health and market stability.In this study,DNA was extracted by three different methods,and an economical,rapid,and high-quality DNA extraction method was selected to meet the subsequent PCR amplification.And quantitative and qualitative detection of pig-derived components in food through fluorescence quantitative PCR and AFLP-PCR technology,and finally established AFLP-PCR method to identify different pig breeds,providing technical basis for the detection of pig-derived components in food,the main contents include:?1?SDS method,guanidine isothiocyanate method and kit method were used to extract animal muscle tissue DNA,and the concentration and purity of the extracted DNA were compared.And design common PCR primers of pig,cow,sheep,chicken and grass carp for common PCR amplification to verify the quality of DNA extracted by the three methods.The results show that the guanidine isothiocyanate method does not require water bath heating,a single sample can be completed within 30 minutes,the extracted DNA OD₂₆₀/OD₂₈₀ ratio is about 1.8,and the OD₂₆₀/OD₂₃₀30 ratio is greater than 2.0.The fresh and high-pressure tissue DNA extracted by the guanidine isothiocyanate method has high DNA concentration and purity,and the product bands in the PCR amplification detection are complete and clear,which fully meets the requirements for PCR amplification.?2?In order to establish a PCR method for the detection of pig-derived components in meat products,the pig mitochondrial cytochrome B gene sequence was compared,and the pig mitochondrial cytb conserved region was selected to design and synthesize pig-specific primers.SYBR Green quantitative fluorescence PCR reaction was conducted with cow,sheep,chicken and duck DNA and pig DNA of different concentrations,which proved that this primer was species-specific.SYBR Green quantitative fluorescence PCR reaction was conducted with cow,sheep,chicken and duck DNA and pig DNA of different concentrations.It proved that this primer was species-specific,and the detection limit could be as low as 5×10-5 ng/?L.And design?-actin universal internal reference primer.Build pig source sex composition containing 0-100%mixed the standard curve of meat,2-??Ct value and pig source sex composition content is a significant linear relationship?R2=0.993?,to the pig source sex composition content is between 0%and 75%of mixed specimens detection,recovery rate of 100.51 to 105.36%.Six commercial samples were tested using the established PCR method,of which the pig-derived content in ham sausage was 4.69%.The established SYBR Green real-time fluorescence quantitative PCR detection method is easy to operate,strong specificity,and reliable data,which provides technical support for the detection of pig-derived components in meat products.?3?EcoR I and Mse I restriction enzymes were used to double digest the porcine genomic DNA and ligate it to the adaptor.Then EcoR I-Mse I preamplification primer pairs were used for preamplification.Finally,from 64 pairs of selective+3 EcoR I/Mse I primers,e-acc/m-ctt combination primers with good repeatability,small number of bands and clear bands were selected to distinguish porcine components,and 4 pairs of selective amplification primers with good repeatability and high polymorphism were selected to identify different breeds of pigs.The results show that in the identification of pig-derived components,the selected primer pairs can distinguish cattle,sheep,chicken,duck and pigs well.The highest similarity between beef DNA and pig DNA in fresh meat was 51.7%,and the similarity between beef and pork DNA in high-pressure meat was 75.1%.In the sensitivity test,pig DNA was added to the DNA of cattle,sheep,chicken,duck fresh and high-pressure tissue according to different concentrations.Analysis of the electrophoresis spectrum showed that the E-ACC/M-CTT combination in fresh and high-pressure mixed DNA the lower detection limit of the primers for detecting pig-derived components was 0.1%.In the variety classification,these four selective amplification primer pairs produced a total of 802bands,with an average of 200 bands per primer pair,and the recognition probability of each primer pair was 100%.All samples were divided into two groups,with an average similarity coefficient Spain Iberian breed of 0.62,while the average genetic Taihu pig and Tibetan pig similarity coefficient was 0.72.In addition,analysis of the ability to distinguish between different pairs of primers three breeds of pigs using PLS-DA,consistent with the results of cluster analysis.