Application Of MtDNA And TYR Gene For Identified Detection Of Mink,Fox,and Raccoon Dog Tissues

Minks,foxes,and raccoon dogs are important fur economic animals.In recent years,they have been raised in large numbers due to their high economic value.In addition to being raw materials for the fur processing industry,carcasses produced after peeling are often processed into livestock and poultry products illegally due to their low prices,which poses a serious threat to food safety.Therefore,the establishment of molecular identification and detection technology for fur animal tissue is of great significance for maintaining the healthy development of the fur animal industry.The purpose of our study is to use the Cytb gene to detection of mink,fox,and raccoon dog tissues from common meat products.Also,The TYR and 12 S rRNA genes were used to distinguish different species of mink.Finally,differences in the D-loop region in mtDNA were used to identify different individuals of the same species of minks.Firstly,this study designed three pairs of specific primers based on the Cytb gene sequences of mink,fox,and raccoon dog published by GenBank,respectively for PCR amplification and enzyme digestion verification,and analyzed the specificity,sensitivity,and intersections of each primer.At the same time,the annealing temperature was optimized and multiple PCR was constructed,and its specificity,sensitivity,and stability were verified by experiments.Secondly,the TYR gene and 12 S rRNA gene in mtDNA of mink were selected for gene amplification,recovery,and sequencing of three different species of mink,and the results were compared and analyzed.Thirdly,the full-length D-loop region of mink mtDNA was selected to design the primers for PCR amplification,cloning,and sequencing,followed by data analysis.The results showed that :(1)The multi-PCR method could specifically amplify the target bands of mink 292 bp,fox 859 bp,and raccoon dog 561 bp.The sensitivity reached 1.0 pg/L.(2)The three different minks of short-haired black,red-eye white and iron-gray minks,were amplified by primers designed by TYR and 12 S rRNA genes.Alignment of the sequencing results with the reference sequence found that the red-eyed mink TYR gene had a missense mutation at site of 137(TGT ? TGA),and the iron-gray mink 12 S rRNA gene had a missense mutation at site of 883(CTG ? CTA).And the combination of TYR and 12 S rRNA gene mutation sites can distinguish three species of short-haired sable,red-eye sable,and iron-gray mink.(3)The full-length amplification of the D-loop region is 1184 bp.The sequencing results show that the A + T base content is the highest,which is consistent with the base composition of the D-loop region of vertebrate mtDNA.Among them,the individual difference between the short-haired black minks is 0.2%-9.5%,the individual difference between the red-eyed white minks is 0.1%-4.7%,and the individual difference between the iron-gray minks is 1.1%-5.0%.The results showed that :(1)A multiplex PCR method was successfully established for the simultaneous detection of mink,fox and raccoon dog tissues,with high sensitivity and stability;(2)The TYR gene and 12 S rRNA gene regions were used to distinguish three different species of mink: black mink,red-eyed white mink,and iron-gray mink.(3)The different individual between minks of the same species can be identified through the D-loop region in mtDNA.