| In recent years,food fraud has become an increasingly prominent issue worldwide.Meat adulteration is particularly serious.The European of "horse meat storm" has been outbreak that some horse meat detected in beef.In China,there is also the phenomenon that the manufacturer mixed cheap meat into high-priced meat,in order to obtain huge profits of the economically motivated adulteration.In the meat industry,ovine is an important pillar industry of animal husbandry with robust consumer demand in China.However,there are some adulterations about high prices of sheep with low price meat of chicken,duck and pork meat.The emergences of these problems seriously interfere with the livestock industry,restricting the improvement of meat quality,and seriously damaged the interests of consumers,and even religious issues and so on.Therefore,it is of great practical significance to improve the authenticity of ovine meat production.At present,the determination of authenticity of ovine is mostly in the relative quantitative stage;method cannot directly obtain the copy number of target gene of species,which affects the accuracy and stability of quantitative results.These cannot be used as a standard method for quantitative testing to popularize,cannot carry out a strong punishment of the criminals in EMA events.Therefore,it is urgent to strengthen the quality detection of meat products and develop the accurate and stable method for quantifying adulterants from ovine and other species.This paper studied on the method for authenticating sheep and quantifying the adulteration's content by droplet digital PCR.The specific studies are as follows:1.In order to accurately detect the adulterant ingredients in sheep,and lay a foundation for the precise quantification,the single copy nuclear genes were selected as the object,and optimized the primers,probes and the system of digital PCR,the detection analysis of sheep and adulterant species was established.The detection result showed that the method can detect species steadily,and the limit of detection is 0.1%(w/w).2.In order to accurate quantitative the pure meat production,the multiplication factors of the ratio of two copy numbers of unit mass of species were obtained by theoretical deduction and experimental verify,and it can be used to transform the copy numbers of gene to the mass of meat.The limit of quantification is 1%(w/w).The results showed that the accuracy and stability of the quantitative method are not affected by the conditions such as meat parts,ultra-high pressure,heating and freezing treatment.3.In order to accurate quantitative the meat products with some ingredients and additives,the standard sample was prepared by adding the internal standard(turkey)meat,and the standard curve was drawn to calculate the transformation formula of the copy number and weight of meat.The limit of quantification is 1%(w/w).The results showed that the accuracy and stability were not affected by the types of additives and ingredients,meat parts,ultra-high pressure,heating and freezing treatment.4.In order to quantitative detection of multiple species adulterate into ovine,two multiple quantification system based on multiplication factor were established.The system can quantify the chicken,duck and pork content in sheep simultaneously,and quantify the fox,mink,and raccoon dogs content in sheep simultaneously.We also established two quantification systems by adding internal standard(turkey)to calculate the species weight,and also can quantify the common and uncommon adulterations in ovine.In summary,this paper uses two kinds of droplet digital PCR technology to achieve a variety of species adulterated in sheep qualitative screening,and single adulterated components of the precise quantitative detection and a variety of animal-derived ingredients at the same time quantification.The representative characteristics detection technology not only quantify the sheep but also to other species.The study is useful for food administration laboratories and law enforcement agency. |
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