Studies On Quantitative Detection Methods For Animalderived Ingredients In Foods By Digital PCR

As China's consumption of meat and meat products grows year by year,the situation of meat products adulteration has become more and more serious.It does not only disturb the economic order,but also endangers people's health.Pig and cattle are sold at a high price and are easily adulterated;Turkey and camel are low yielded which leads to high prices and the unbalance between supply and need,resulting in the fact that they also encounter adulteration.Besides,Fox is an important breed which provides fur in China,with a large demand of fur,their by-product meat has been found many times as fake mutton and fake beef in the food market.These counterfeit and doping problems have seriously harmed the benign development of China's meat industry,restricted the quality improvement of meat and meat products,severely damaged the legitimate interests of consumers and even led to religious conflicts.Therefore,improving the authenticity detection method for pig,cattle,turkey,camel,and fox is of great significance in protecting the food safety of residents.At present,the authenticity identification method for pig,cattle,turkey,camel,and fox is mostly qualitative and semiquantitative,which cannot determine whether the detected components are intentionally or unintentionally falsified.Therefore,it is urgent to establish a specific,stable,sensitive and universal quantitative method which can precisely detect pig ingredient,cattle ingredient,turkey ingredient,camel ingredient and fox ingredient.Based on digital PCR technology,the current study has established a qualitative and quantitative detection method for pig,cattle,turkey,camel and fox,which can cover droplet digital PCR(dd PCR)and chip digital PCR(cd PCR).The specific research procedures are as follows:1.We chose single-copy genes from the genome,designed and selected five pairs of special primer and probes and respectively established a detection of pig ingredient,cattle ingredient,turkey ingredient,camel ingredient and fox ingredient,suggesting that the limit of detection of copy numbers concentration of the five species is lower than 1.2 copies/?L and the sensitivity can reach the degree of 0.01%.2.We selected the Promega genomic DNA extraction kit to ensure the DNA extraction effect.By measuring the water loss rate of different tissues and organs,we explored the quality of dry powder of different tissues and organs,finding that the rate of water loss varies in different tissues and dry powder quality-DNA concentration is also the case.Therefore,the subject of quantitative studies selected muscle tissue.Finally,a quantitative PCR detection method for pig ingredient,cattle ingredient,turkey ingredient,camel ingredient and fox ingredient was established,suggesting that the limit of quantitation of copy numbers concentration of the five species is lower than 7 copies/?L.We explored the linear relationships between copy numbers concentration and fresh meat for pig muscle,cattle muscle,turkey muscle,camel muscle and fox muscle in dd PCR and cd PCR.3.We prepared the simulated samples of different percentages,the mixture of two kinds of animal ingredients and the five kinds of animal ingredients,and quantitatively detected the quality of simulated samples and commercial samples.The results indicated that the simulated samples with mass content of 5% or above are similar to the true values,and the recovery rate was between 80% and 120%.The results of commercial samples showed that the established digital PCR quantitative detection method can be effectively applied to the detection of meat samples.In summary,based on the digital PCR technology,the qualitative and quantitative detection methods which are established in the current study can realize a more precise detection for pig ingredient,cattle ingredient,turkey ingredient,camel ingredient and fox ingredient in mixed samples.It is proved to be helpful in providing market surveillance for meat and meat products with technical support.The study also provides insights to the follow-up researches which also focus on quantitative detection method for meat product.