DNA-Barcoding Technique For Identification And Detection Of Components Of Animal Origin

Improve the quality of life standards, meat production reached a number unprecedented in human history. Meat adulterate behavior,the spread of animal diseases,as well as the religious taboo against meat rare animal protection and other issues, has been deeply concerned about the current society.The use of PCR technology to identify the source of animal-derived ingredients is the scientific basis for the fight against illegal activities,but also a strong guarantee for the protection of rare animals.Through genetic biological methods,and strive to animal-derived ingredients from discriminating between different species and between different species.Also the use of precise and rapid PCR technology features for qualitative and quantitative detection of animal-derived ingredients and content.The experimental study is divided into three parts:(1) Barcoding technology is based on the International Barcode of Life Alliance (CBOL,the Consortium for the Barcode of Life) proposed a sequence determined by the area.By NCBI query and Oligo6.0 and Primer5.0 screening software,has been universal primers CO Ⅰgene can be amplified gene fragmentCO Ⅰ cattle,sheep,pigs,poultry,rabbits,dogs and cats seven species.Vector NTI.11.5 then use software within Afa I screening test as the restriction enzyme.The results show that the cut digestion of cattle,sheep,pigs,poultry,rabbits,dogs and cats can be Afa I restriction of seven species into different fragments,which identify different species of animal-derived ingredients.(2) According to sources conserved gene sequences of mtDNA CO Ⅰ and moderate evolutionary characteristics,designed Realtime-PCR melting curve method to detect pig-derived ingredients that speed up the detection rate,but also increases the number of simultaneous detection of samples.Meanwhile,in order to avoid the impact of food PCR inhibitors,the test set-GCG 125bp gene fragment was purified as an internal reference,control of false negative results.Optimized PCR conditions,and specific product GCG pigs were gene-specific and a melting peak at 79.2℃,75℃.Set cattle,sheep, poultry,rabbits,rats and control,no specific amplification.The sequencing results showed that,PCR product length of the primers specific for porcine 245bp,the nucleic acid sequence corresponding to the sequence in NCBI anastomosis. LOD value of 0.001% for the method.(3) the use of’functional gene differences between different varieties to identify the source of animal-derived ingredients.First,we need to select a highly conserved genetic Genetic this experiment chose WZSP (WZSP) and Duroc as the identification of objects,through literature and the general body learned WZSP pigs have multiple SNP loci in the GHR gene,There are two SNP mutation sites are intentionally and changes the conformation of the protein, influence the effect of growth hormone. GHR gene sequence is based on a porcine species query NCBI SusCrafa obtained according to the reported primer. Amplification WZSP and Duroc GHR gene sequences obtained product,then the Vector NTI.11.5 software filters use the information endonuclease Pst I Experimental results show that,after endonuclease digestion with restriction enzyme Pst I WZSP GHR gene sequence of the amplification product will be with two,while the ordinary figure appears a strip Duroc pigs,indicating that the method successfully identified animal origin ingredients from different sources varieties.By mixing test,the method detection limit (LOD) was 5%.High LOD value may be due to a functional protein gene compared with mitochondrial genes in the individual cells on a much smaller number of genes,and longer GHR gene sequence to degradation fracture.The main purpose of this experiment was to establish the use of DNA-Barcoding technology simultaneously identify different species of pig-derived ingredients derived ingredients and batch testing methods.Then,based on the identification of the species,the use of SNPs of GHR gene to identify species WZSP derived ingredients.But also lay the foundation for the development of testing standards of accreditation bodies,provide the basis for future animal-derived ingredients detection technology innovation.