Identification Of Species In Meat Products Using PCR-RFLP Assay

This paper investigated the use of PCR(polymerase chain reaction) and RFLP(restriction fragment length polymorphism) for identification of the origin of beef,pork,mutton,chicken in their products. PCR amplification yieled relative species-specific fragments.The amplicons were digested with restriction endonuclease.Therefore,the aim of this study was to set up a PCR-RFLP method to trace animal species in processed meat products.This paper includes four parts:Identification of bovine-derived materials in meat products: DNA was extracted from 7 raw kinds of animal meats(pork,beef,mutton,chicken,duck,fish and rabbit meat) and 4 kinds of meat products (beef charqui,beef jujube,beef ham sausage and autoclaved beef). A set of universal primers of Cytb gene was taken as an endogenous reference gene to check the quality of template extraction.Then raw beef was set as positive control, 4 kinds of beef products as testing sample, other 6 kinds of animal meats as negative control.Bovine-specific primers to detected bovine derived materials in 11 kinds of samples,it showed no cross-reactivity with DNA from other species. In order to confirm the specific amplification of the expected products , Mbo I digestion assays was performed. This method was adapted to commonly identify bovine-derived materials in food products.Identification of porcine-derived materials in meat products: DNA was extracted from 6 raw kinds of animal meats(pork,beef,mutton,chicken,duck and rabbit meat) and 7 kinds of meat products (autoclaved pork,pork ham sausage,pork jujube,dried pork floss,beef charqui,beef jujube and beef ham sausage).PCR-RFLP of the mitochondrial cytochrome b gene for PCR amplification was used to yield 359 bp fragment in each of these species. Alu I digestion of amplicons allowed specific recognition of pork(244 bp and 115 bp fragments)and beef(190 bp and 169 bp fragments),whereas amplicons from other species were not cut. So, pork can be detected,and the amplicons were digested resulting in a pattern that could identify and differentiate the pork and beef.Identification of ovine -derived materials in meat products: DNA was extracted from 7 raw kinds of animal meats(pork,beef,mutton,chicken,duck,fish and rabbit meat) and 4 kinds of meat products (boiled mutton, autoclaved mutton, fried mutton, roast mutton). A set of universal primers of Cytb gene was taken as an endogenous reference gene to check the quality of template extraction.Then raw mutton was set as positive control, 4 kinds of mutton products as testing sample, other 6 kinds of animal meats as negative control. Ovine -specific primers to detected sheep-derived materials in 11 kinds of samples,it showed no cross-reactivity with DNA from other species.In order to confirm the specific amplification of the expected products , Sau3A I digestion assays was performed. This method was adapted to commonly identify ovine -derived materials in food products.Identification of chicken-derived materials in meat products: DNA was extracted from 7 raw kinds of animal meats(pork,beef,mutton,chicken,duck,fish and rabbit meat) and 4 kinds of meat products (chicken ham sausage, autoclaved chicken, soy stewed chicken wing, roast chicken). A set of universal primers of Cytb gene was taken as an endogenous reference gene to check the quality of template extraction.Then raw chicken was set as positive control, 4 kinds of chicken products as testing sample, other 6 kinds of animal meats as negative control.Chicken-specific primers to detected chicken-derived material in 11 kinds of samples,it showed no cross-reactivity with DNA from other species.In order to confirm the specific amplification of the expected products , Hind III digestion assays was performed. This method was adapted to commonly identify chicken-derived materials in food products.