The Research And Development Of Kit For The Genes Detection Of Six Livestock Meat

With the improvement of living standards and changes in consumption attitudes,the demand for meat products is increasing.Under the drive of interests,many traders adulterate cheap meat into high-priced meat and adjust the taste and color of products by using additives,to achieve a real effect,so as to increase business profits.Especially for those processed products such as beef,mutton and lamb skewers,It is difficult for consumers to screening by texture and taste.The arise of these adulterated meat products will seriously affect the health of consumers.Therefore,the establishment of a precise,rapid and high-throughput detection method,to provide a strong technical support for the supervision of animal-derived foods is an important issue that needs to be resolved urgently.Based on the Species-specific PCR technology,a qualitative detection method for adulterated meat from six livestock breeds:cattle,sheep,pig,dog,horse and donkey was established,a test kit was developed in this paper.The results are as follows:1 In order to select the best method for rapidly extracting DNA from animal muscle tissue,beef was used as the test material,four kinds of pre-treatment methods(common blast drying,vacuum freeze-drying,liquid nitrogen grinding,meat grinder mincing(blank control))were combined with seven extraction methods(including SDS method,guanidinium isothiocyanate method,CTAB method,chloroform-sodium acetate method,KI method,phenol-chloroform method and Tiangen kit method)to extract beef DNA,The trace nucleic acid protein concentration analyzer,agarose gel electrophoresis and Species-specific PCR amplification were used to detect the integrity,concentration,purity and PCR amplification adaptability of the extracted DNA fragments.The results showed that for the sample pretreatment,the liquid nitrogen grinding method can effectively prevent DNA degradation caused by heating during the grinding process,thus the integrity of the DNA can be ensured and the sample pretreatment time can be saved;among these seven kinds of DNA extraction method,the DNA of bovine muscle tissue extracted by the SDS method and the Tiangen kit method can meet the requirements of subsequent molecular biology experiments such as PCR,but the SDS method is superior to the Tiangen kit in DNA purity,concentration,and stability.2 The annealing temperature is a key factor which significantly affecting the success of PCR amplification.In order to amplify clear target band while avoiding the influence of miscellaneous band on the determination of the test results,the optimal annealing temperature for each pair of primers was explored in this experiment.According to the order of cattle,sheep,pig,dog,horse and donkey,the annealing temperature are 54℃,65℃,62℃,46℃,67℃ and 66℃,respectively.3 The sequencing results of PCR products were compared with the sequence of the target gene,the results showed that the identity of the sequencing result of PCR products of cattle,sheep,pig and donkey with the target gene sequences in Gen Bank were 100%,100%,96%and 99%,respectively,which further proved that the preparation of the recombinant plasmid was successful.While after repeat research,the sequencing of the recombinant plasmids of dog and horse showed that they have high homology with the target genes of argali and tarsier monkeys.May be owing to the use of ordinary Taq DNA polymerase in the process of plasmid preparation makes the gene mutation,high-fidelity enzymes will be used in the future work to minimize the probability of mutations.4 Based on the foregoing research,the ultimate goal is to develop a PCR kit,which includes two sections,for DNA extraction and further detection,respectively.The DNA extraction section is an new extraction method which combines the SDS method with the adsorption column in the Tiangen kit.This method integrates the advantages of the SDS method and the Tiangen kit,with better concentrations,purity,DNA integrity,and PCR amplification adaptability.In the previous experiments,the species detection conditions have been optimized,so it’ s convenient for purchasers to get the detection results within 2.5 hours as long as the DNA of samples are extracted according to the instructions.The test costs of the kit is calculated by double counting for each sample,which is approximately to be 30 RMB/sample,compared with those existing kits and detection methods,the cost of this kit is lower,and the detection sensitivity is higher,which is conducive to the promotion in the later period.In summary,the PCR kit developed in this study has the advantages of high sensitivity,low cost and can simultaneously detect multiple samples.The method can fully be used for the detection of processed meat products,providing the method guidance and technical support for the food inspection agencies,which is of great significance for the rapid detection of livestock-derived components in foods.