Research On A New Method Of Molecular Biology For The Detection Of Adulterated Meat

Food safety has become a great concern of the whole society,and the meat adulteration events happened quite a lot.Because of the complicate of the source and deep processing,it is difficult to detect the qualitative and quantitative of the adulteration of meat and meat product.These also brought great challenge to the government in the maintenance of market order and assurance of trade fair.At present,the spices identification technology which can analyze and divide the specific source of animal meat in food sample based on molecular biology method has developed rapidly.Even so,in the qualitative and quantitative detection of product mixed with several kinds of unknown meat according to the recent standard and method,it has difficulties and will consume quite a long time for the lack of primer.For the high copy number,simple structure of genome and easy to purification of mt DNA(mitochondrial genome),it can be used in the accurately detection of species through the compariion between the result of mt DNA sequencing to the open-access database.With the help of the new high-throughput sequencing technique,it is possible to test several sequences in one sample at the time.What's more,this method can identify the source of animal meat with the coverage up to 99.94%.So it has a broad application prospect in the qualitative analysis of complicated samples.Real-time fluorescence quantitative PCR is an advanced nucleic acid quantitative technique with high sensitivity,which is based on the original PCR technique.It has been widely applied in the detection of genetic modified food,rapid detection,and molecular diagnostics and so on.In the market surveillance,to identify whether the sample is polluted unintentionally or adulterated intentionally,we need a better qualitative detection method.Firstly,the paper established a procedure of extracting mt DNA from meat and meat products.Secondly,built a qualitative analyzing method in order to amplify the extracted mt DNA characteristically.After that,through the high-throughput sequencing combining with the database analyzing we can get the qualitative results.Then the quantitative analysis method was established by fluorescence quantitative PCR and quantitative standard curve,we can obtain the relative quantitative results of samples.Further considering the quantitative results related to DNA extraction efficiency and quality,this paper explores the effects of microbiological and meat preservation time on DNA extraction efficiency and fragment size.The main findings are as follows:(1)The mt DNA was extracted through phenol extraction CTAB(cleavage method),phenol extraction SDS(cleavage method),Na Ac-phenol extraction CTAB(cleavage method),Na Ac-phenol extraction SDS(cleavage method).The result was compared through quantitative analysis and showed that the higher content of mitochondrial DNA in DNA samples extracted by Na Ac-phenol extraction CTAB(cleavage method)was more suitable for qualitative and quantitative analysis.(2)The Cyt B gene(about 400bp)in the extracted mitochondrial DNA was amplified by PCR.The amplified product was subjected to high-throughput sequencing.And the qualitative analysis of the meat components in the samples was carried out with an open database.In order to compare the difference of sequencing quantity and precision,the total DNA of the sample was randomly divided into two groups: the procedure of high-throughput sequencing was compared with the two procedures,the one taken in this paper had an advantage of lower coast and higher quantities of sequencing with 100% accuracy rate.(3)The relative quantitative analysis of pork components in different proportions of cattle-pig mixed meat samples(100%,80%,60%,30%,15%,1%)was carried out by fluorescence quantitative PCR.The(1%,0.5%,0.25%,0.1%,0.01%)of the simulated samples were tested for the sensitivity of the study method.The standard curve of the logarithmic value lg(x %)of the percentage of pork in the standard sample of cattle-pig mixed meat was established as the abscissa of the difference between the pig's target gene and the Ct value of the internal reference gene.The results showed that the method established in this paper had a good linear relationship with the pork content of 1% ? 100%(the correlation coefficient R2> 0.985),and the detection limit was 0.1%.(4)In this paper,we studied the effects of psychrophilic bacteria(Pseudomonas,Acinetobacter and Lactobacillus),storage time on DNA extraction efficiency and fragment size.The results showed that the extraction rate of total DNA and the size distribution of fragment were significantly affected by different storage time.With the longer storage time of fresh beef,the smaller the DNA fragment,the ratio of DNA(A260 / A230 represents the degree of organic compound contamination)varied from 2.02 in 1d to 1.01 on 14 d,(A260 / A280 for protein contamination)ratio varied from1.98 in 1d to 1.66 in 14 d,and the extraction efficiency decreased(0.0202% to 0.0006%).