An Approach For The Identification And Quantification Of Goat And Sheep Meat Using Multiplex PCR And Real-time PCR

The study is aim to establish an assay to identify goat and sheep meat in qualitative and quantitative way, respectively. To identify the goat and sheep meat, attempts were made to establish one-step multiplex PCR assay for the identification of goat, sheep, mink, nutria and duck. Primers were designed from mitochondrial DNA and generated specific fragments of 577 bp for goat, 265 bp for sheep, 231 bp for mink, 812 bp for nutria and 351 bp for duck, respectively. Specificity of five primer sets was confirmed bythe DNA offive animal species. No interference effect was observed between primers and DNA templates. Through the optimization experiments, the optimum proportion of primers is goat: sheep: mink: nutria: duck=3:5:4:3:2, and the optimum annealing temperature for the multiplex PCR is 59℃.Taqman real-time PCR assay using species-specific primers and probestargeting the mitochondrial Cyt b gene and universal eukaryotic primers amplifying a conserved fragment of the 16 S r RNA gene has been developed for the detection and quantification of goat and sheep DNA in meat mixture. Standard curves were established using series of gradient-diluted DNA, and the relationships between mass and DNA concentration of each species(D values) have been determined. The assay was tested on meat mixtures consisting of different kinds of species and concentration. The results showed that the mean absolute error of detection was 6.82% for goat meat, and 8.14% for sheep meat.The sensitivity was detected for both multiplex PCR and Real-time PCR. Results showed the detection limit is 0.1ng formultiplex PCR and 0.01 ng for real-time PCR. Both assays can be used to the detection of fresh, boiled and autoclaved meat. Meat mixed with corn was detected using multiplex PCR, and no significant effect was observed.The major steps of the detection and quantification for meat mixtureswhich contain goat or sheep meat are:(1) extract DNA of unknown sample;(2)identify meat species using multiplex PCR assay;(3) determinate D values of each species;(4) establish the standard curve;(5) amplify the real-time PCR;(6) measure the content of goat or sheep meat.