Study On Identification And Inspection Of Meat And Meat Products In Animal Derived Food

In recent years, the animal origin foodstuff adulteration, adding various chemical additives excessively, the use of non-food raw materials and ingredients and so on various food safety issues have appeared constantly. Therefore, establishing a rapid and accurate detection method, identification of animal-derived ingredients for food has become a problem of concern.This experiment has carried out multiplex PCR technology to identify animal origin ingredients of meat foodstuff by means of molecular biology. With the characteristics of the polymerase chain reaction, we study on the specific genes of various types of meat and meat products to achieve qualitative and quantitative detection for the corresponding animal origin meat and meat products.By using five extraction methods (the KI method, Chloroform - sodium acetate method, Phenol and Chloroform method, Kit and FTA filter method ), genomic DNA extracted from fresh animal tissue was detected through 1% agarose gel electrophoresis ,the results show that the band is bright ,neat and of good integrity with a clear background .Use the same sample, KI method and chloroform-NaAc method received the best effect, kit method take second place. Ultraviolet spectrophotometer measured that OD260 nm/OD280 nm were in 1.70~1.93, the concentration was 0.1~2.0μg/μL, DNA was not polluted by RNA and protein or phenol.The PCR reaction system and reaction procedure were determined through optimizing Taq DNA Polymerase, dNTPs, Mg2+, primers, circulate parameters, annealing temperature and time. The normal PCR reaction system of duck and pork (primer1) was: 25μL blends liquid, the concentration of each primer was 0.2μmol/L at last, 0.5μg template, add ddH2O to 50μL.The reaction procedure of duck was: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 56℃for 40s, extention at 72℃for 60s, run 35 cycles, final extention at 72℃for 5min.The reaction procedure of pork was: pre-denaturation at 94℃for 1min, denaturation at 94℃for 60s, annealing at 54℃for 30s, extention at 72℃for 30s, run 35 cycles, final extention at 72℃for 5min.after the amplification, the bright bands of Duck DNA appeared in 226bp position, pork DNA in the 149bp position , they were the same size with the aim bands , blank control produced nothing and of no non-specific bands. The lowest detection limit of KI method was 0.005%, Chloroform-NaAc method was 0.01%,Kit method was 0.1%.The double PCR reaction system of duck and pork (primer1) was: 25μL blends liquid, 2μL duck primer, 0.5μL pork primer(the concentration of each primer was 20μmol/L ),1μg duck template, 0.5μg pork template, add ddH2O to 50μL.The reaction procedure was: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 56℃for 40s, extention at 72℃for 60s, run 35 cycles, final extention at 72℃for 7min.Two distinct bands were amplified simultaneously, the position was the same as the aim bands.Multiplex PCR system can be established with the specific primers of cattle, goats, sheep, pigs, chickens, horses, deer and rabbit.get segment size of PCR amplification products was different because of different species. The reaction system was: 25μL blends liquid, 6.5μL mixed primers, 0.5μL primer, add ddH2O to 50μL. The reaction procedure was: pre-denaturation at 94℃for 4min, denaturation at 94℃for 30s, annealing at 58℃for 30s, extention at 72℃for 30s, run 35 cycles, extention at 72℃for 4min. After repeated tests, when the mixing proportion of primers is common primer: cattle : goats : sheep : Pig2: Chicken: horse: deer : rabbit = 1:2:3:0.5:1:0.6:2:1.5:1.8 (1 means 20pmol/50μL ) , the amplification effect is most ideal. This method can amplify eight specificity bands , the examination sensitivity achieves 0.01%.The test results of the actual sample showed that, the mixed primer is fully capable of amplification of specific DNA fragment from different kinds of meat products, and can be fully applied to cooked meat products to the judgment of the authenticity and adulterated ingredients .The method established by this research is specificity , high sensitivity, fast and easy and so build a technology platform for the rapid detection of animal origin ingredients in food, and can be application and dissemination as a recommended method by inspection departments.