Quantitative Detection Of Chicken-derived Components In Mutton By Real-time Quantitative PCR

In recent years,there have been frequent cases of adulteration of meat products around the world.The main adulterating method is to incorporate some or all of the cheap meat into the high-priced meat products,and not to indicate the ingredients in the product label.In order to protect the interests of consumers,many countries and regions have enacted regulations concerning the labeling of meat products and established many animal species identification methods based on DNA or proteins(polypeptides)to comply with the implementation of corresponding regulations.However,in the current national standards,industry standards or literature reports,the detection of animal species composition in meat products mainly focuses on qualitative or semi-quantitative detection.With the continuous changes of adulteration methods,the key to effectively distinguish between unintentional pollution and illegal addition is to determine the proportion of adulteration.In China,On the market,they are many faked mutton made by mixing pork,chicken,duck,etc with sheep fat or adding only mutton powder,essence into the meat.Therefore,there is an urgent need to establish a stable,accurate,and effective method for the quantitative detection of the percentage of chicken meat in mutton,which provides a strong scientific basis for quality inspection department to quantitatively inspect the meat components.Based on the real-time fluorescent PCR technology,this paper established a quantitative detection method for broiler-derived components in lamb,the main contents are as follows:(1)Study on the optimum method for drying moisture in lamb and chicken samplesIn this paper,the moisture of the mutton and chickensample was treated by direct drying method and vacuum drying method,and an effective sample preparation method was determined.The results showed that the direct drying method had the shortest treatment time and did not affect the quality of the sample nucleic acid.It was determined as the best method for moisture drying of lamb and chicken samples.(2)Design of specific primers and probes for goat and chicken-derived componentsIn this paper,two pairs of specific primers and probes were designed using a single copy gene for sheep and chicken,and a real-time fluorescence PCR quantitative detection system was successfully established.The results showed that the designed primers and probes have good specificity,and no reaction occurs with the other components except the target species.The annealing system optimizes the amplification of the detection system.(3)Study on the best extraction method of animal tissue genomic DNAIn this paper,a rapid,stable and high-quality method for the extraction of genomic DNA from animal meat tissue was established based on the methods of DNA extraction from four commonly used animal tissues.The results showed that the mutton and chicken genetic DNA extracted by magnetic bead method and automatic nucleic acid extractor had the best quality,the highest stability and the shortest extraction time.(4)Quantitative detection of lamb and chicken ingredients by real-time fluorescence PCRIn this paper,we used real-time fluorescence PCR and digital PCR to explore the relationship between the quality of mutton and chicken and DNA content,gene copy number,and the relationship between DNA content,gene copy number and Ct value.Differences in mathematical models establish a stable,accurate,and effective method for the quantitative detection of the percentage of chicken meat in mutton.The results show that based on the mathematical relationship between DNA content and gene copy number,all of them have a good linear relationship,and R~2 is more than 0.98.(5)Quantitative detection of chicken-derived components in mutton by real-time fluorescence PCRIn this paper,the use of simulated mixed samples and 34 commercial lamb samples verified the practicability of the established quantitative methods.The results show that the mathematical model established based on gene copy number has better quantitative stability and accuracy.The detection limit can reach 5%(w/w),which is effectively applied to the detection of the actual faked mutton sample.In summary,the real-time fluorescent PCR quantitative detection method based on gene copy number can be used to quantitatively detect the composition of adulterated chicken in mutton.It can be used as a basis for correctly identifying intentionally adding and unintentional contamination,and providetechnical protection for law enforcement agencies.