The Establishment Of Animal-derived Ingredients Comfirmed And Components Quantified System In Meat And Meat Product

Meat and meat products as an important part of people's diet,the misleading labeling and the addition of fraudulent ingredients of meat products are the major problems facing meat product today.In this study,a rapid screening multiplex PCR method for detect the heterologous animal components in meat and meat products was established,provided scientific basis for relevant departments to monitor the adulteration of meat and meat products.And a complete quantitative real-time PCR method was established for detect the cattle-derived components in meat and meat products,to provide reference for the study of quantify cattle-derived components in meat and meat products.The results of the study are as follows:1.According to GenBank,the cells pigment b(Cytb)gene of mitochondrial DNA from beef(Bos taurus),sheep(Ovis aries),pig(Sus scrofa),chicken(Gallus gallus),duck(Anas mallard)and horse(Equus caballus),design and synthesis of specific primers.By optimizing PCR reaction conditions and reaction system components,established a five-fold PCR detection system based on cattle and sheep,the reaction conditions of this method can effectively amplify the target fragments of five species,and the band is clear,indicating that there is no complementary pairing between the primers.This method can screen heterologous animal components in meat and meat products.In line with the national standard test results,test results with high accuracy and credibility.2.Using bovine mitochondrial DNA cytochrome b gene as target gene,Optimized design of specific primers and Taqman fluorescent probes,and optimized reaction conditions and systems,established a quantitative real-time PCR method for the detection of cattle derived ingredients in meat products.Amplification showed a typical "S" curve,expansion of the index significantly,the specificity reaches 100%.The sensitivity of the system can reach 10 pg / ?L.With a short test time,DNA extraction from the sample to the fastest results can be completed within 4 hours.Detection fragment length of 95 bp.Reduce the possibility of can't achieve the purpose of testing,because of DNA chip length reduction during processing of meat products.3.Based on the mitochondrial DNA,a segment of highly conserved sequences shared by animal origin components was screened to determine the reference gene.Taqman quantitative real-time PCR relative quantification method was used,by internal reference genes introducted,achieved the quantitative detection of cattle derived ingredients in meat and meat products,the average recovery was 98.62%,it shows that this method has higher accuracy and credibility.The internal reference primers and probes designed in this experiment have good versatility.The amplification results showed that only the DNA of animal-derived samples was used as a template for fluorescence signal,and other samples had no fluorescence signal,had good versatility and specificity for animal-derived ingredients.