Identification Of Animal Provenance Components In Meat And Products Based On Nucleic Acid Molecular Level

As the old Chinese saying goes:food is everything.Food safety has been livelihood issues and meat adulteration already becomes the issue that common people pays close attention to most.With the complexity of adulterated ingredients and the diversification of flavor and fragrance,it is difficult to identify the animal derived ingredients of meat by the sensory identification,microscopic identification and the ways based on the protein level.At present,adulterated meat occurs frequently because of the lack of standard animal food traceability system for meat,and the imperfectness of the relative law.In order to ensure the safety of consumers,it is urgent to establish an accurate and convenient identification method for the animal derived ingredients of meat products.Based on the molecular level of nucleic acid,the conventional PCR identification method for common animal and poultry meat provenances,the quadruple PCR identification method for doped meat and the real-time fluorescent PCR method for the quantification of sheep and cattle components in meat products were established based on the level of nucleic acid molecules.This study can provide reference for food safety departments to establish standards for the identification of animal-derived ingredients.1.Conventional PCR identification methods for common animal and poultry meat:Aimed at the common meats of pig,cattle,sheep,chicken,and duck,the mitochondrial gene sequences of pig(Sus scrofa),cattle(Bos taurus),sheep(Ovis aries)chicken(Gallus gallus),and duck(Anatinae)were retrieved from GenBank.Based on the gene sequences,five species-specific primers were selected by the design of Primer Premier 5.0 software.The amplified product fragments are 388bp,109bp,399bp,364bp,254bp,respectively.were and the Blast comparison was conducted in the NCBI GeneBank.After the PCR system and conditions were optimized,the amplified products of the five species were sequenced in two directions.The homology of the amplified products was more than 97%by the tool Blast in NCBI GeneBank.The primers were uniquely amplified with clear bands under the template of pig,cattle,sheep,chicken,duck,donkey,fox,mouse and deionized water.It could prove that the specificity of the primers is good.The designed primers were sensitive which could meet the requirements for identifying the five common meat ingredients.The minimum detection limit for DNA template of pig,sheep and duck were all 0.5ng/?L,and the cattle and chicken both were 5ng/?L.Samples of cattle and mutton products from wet marke or supermarket were extracted for testing.Mixing cheap duck meat into beef and mutton for adulteration to reap fabulous profits.2.Quadruple PCR Identification Method for Doped Meat:Four pairs of Omega PCR primers with good specificity were designed and screened with rat,chicken,duck and horse mitochondrial COX 1 as the target gene.After the primer specificity verification,the multiplex PCR system and the optimization of the reaction procedure,a quadruple PCR system was established to identify the four species of rat,chicken,duck and horse at one time.The amplified fragment sizes of the four species were 644 bp,336 bp,512 bp and 204 bp,which were consistent with the target fragment size,and the homology was over 97%.The interference between primers and templates was relatively weak.The system of single,double,triple and quadruple templates was suitable.The sensitivity of the system template was up to lng/L,and the repeatability of the quadruple system was good.The system can achieve the purpose of multi-species detection in the same reaction tube,save time and cost,and be economical and efficient.3.Real-time fluorescence PCR method for quantification of sheep and cattle components in meat products:In order to quantify sheep and cattle components in meat products,specific primers were designed with cytochrome b gene sequence of sheep and cattle,and universal primers were designed with 16S rRNA as a reference gene control for normalization.The specificity,sensitivity and generality of the reference genes were tested.The gene amplification showed a typical "S" curve,which expansion of the index significantly.The sensitivity of the DNA template of sheep could reach 10pg/?L and the cattle reached 0.64pg/?L.The real-time quantitative method had a higher sensitivity for testing.The standard curve was established between the logarithm of the percentage mass of sheep(cattle)and the corresponding ?Ct value.The result showed that the two standard curves had a good linear relationship whose the correlation coefficients were larger than 99%.In order to evaluate the reliability of the standard curve,the recovery rate of mixed sheep(cattle)meat samples with known content was tested.The recovery rate of samples is high which was 93.07%-106.20%.It confirmed that the real-time quantitative method was suitable for quantifying the cattle and mutton derived components detection in meat products.Samples of products from the local market or supermarket were extracted for testing.The result showed that the content of beef and mutton was on various levels.