Studies On Qualitative Identification Methods For Animal-derived Ingredients In Foods

The animal-derived ingredients from pork, beef, mutton and chevon in foods were identified by molecular biology technology such as species-specific polymerase chain reaction(PCR), DNA barcoding, restriction fragment length polymorphism of polymerase chain reaction products(PCR-RFLP), terminal restriction fragment length polyraorphism by universal primer polymerase chain reaction (UP-PCR-TRFLP) to compare advantages and disadvantages of the four methods , and to establishment the qualitative identification methods for animal-derived ingredients in foods. The results were as follows:(1) The tests showed that the gene specific primer designed according to the mtDNA D-loop region and Cytb genes were of high specificity. The any animal-derived ingredients from pork, beef, mutton and chevon in foods could be identified accurately by the specific primer, it also could be used to detect mixed samples.(2)After the COⅠgene had been sequenced by deoxidizing method, the sequencing results were compared by multiple sequence alignment, it was found that the nucleotide difference of COⅠgenes between swine, sheep, cattle are 19%～25.9%, nucleotide differences within species are only 0.1%～1.6%. COⅠgene Contained a large number of single nucleotide mutation sites which can mark species characteristics, so the animal-derived ingredients from pigs, cattle and sheep products in food could be identified accurately by gene barcode technology on bases difference of COⅠgene, but this method was not suitable for the detection of mixed samples.(3)Three specific gene segments of pork, beef and mutton were obtained after COI genes had been identified by Hinf I restriction enzymes among enzyme digestion analysis PCR-RFLP. The lengthes of segments were 40bp, 411bp and 449bp for pork, 46bp, 377 bp and 479bp for beef , 144bp, 376~381bp for sheep respectively. Based on the electrophoresis test results, it was safe to say that PCR-RFLP method could be used for qualitative identification of animal-derived ingredients from pork, beef, mutton in food, but it was not suitable for the detection of mixed samples.(4)A pair of universal primers for beef and mutton was be designed in conservative regions of COⅠg ene with the UP-PCR-TRFLP method . The forward PCR primer was labeled with 6-carboxyfluorescein amino hexy(6-FAM) fluorescent dye and reverse PCR primer was labeled with ROX fluorescent dye after digestion by restriction enzyme HinfⅠand BlnⅠ, The restricition fragment were analysed by capillary electrophoresis using an antomated DNA sequencer. The special peaks of swine and beef were established. The PCR products of beef could appeared 373bp fragments at upstream and 46bp fragments at downstream by the restriction enzyme HinfⅠ. Both of fragments were regarded as the characteristic fragments for beef. PCR products of sheep appeared 515bp fragments at upstream and 203bp fragments at downstream by same method. Both of them were regarded as the characteristic fragments for sheep. There were different electrophoresis peak spectrum from different species. Therefore, UP-PCR-TRFLP method could be used for identification of animal-derived ingredients from beef and mutton products in foods and it was also a detection method for mixed samples.(5) The sequencing results of COⅠgene in mtDNA and extinction enzymes cut analysis showed that COⅠgene contained a large number of base mutation single nucleotide mutations which could be used for identification of species. Those mutation sites were of high distinguish function on species identification of pork, beef and mutton, so they could be used for identification of animal-derived ingredients in animal-derived ingredients...