Establishment Of QPCR And DdPCR For Quantitativedetermination Of Mutton Origin Components In Meat

China has a large population of livestock and poultry meat,but the current situation of adulteration of meat at home and abroad is worrying.Many bad businesses have to maximize the benefits,replace them with cheap meat,inferior meat or partially replace high-priced meat.The purpose,which seriously damages the interests of consumers and also has an adverse impact on market regulation.The current detection methods are mainly qualitative or semi-quantitative techniques,and it is impossible to accurately distinguish between illegal addition and unintentional pollution.The method established in this study mainly uses real-time fluorescent quantitative PCR and micro-drop digital PCR detection technology.Real-time fluorescent quantitative PCR adds fluorophores to the reaction system,and uses the fluorescence signal accumulation to monitor the whole PCR process in real time,and finally passes the standard curve to the unknown template.Perform quantitative analysis.Digital PCR uses the limiting dilution method and the statistical principle of Poisson distribution to calculate the DNA concentration without the need of a standard curve,and is not affected by changes in PCR amplification efficiency in different samples and experiments,and can achieve as low as a single copy of the nucleic acid molecule.Absolute quantification.The internal standard species and sample added in the method are simultaneously pretreated,extracted and PCR amplified,which can effectively reduce the influence of objective factors on the results.In this study,real-time quantitative PCR and micro-drop digital PCR detection techniques were used to establish a quantitative detection method for sheep-derived components.The main results are as follows:(1)The single-copy gene of sheep and bovine-derived components was selected for primer design.By homologous alignment of genome sequences of multiple species,specific conserved regions of target species were selected and designed separately.Sheep,bovine-derived specific universal primer probes,and condition optimization of the reaction system.The two pairs of primers and probes designed have good specificity,and only react with the target species,no non-specific amplification occurs,and the detection system has a good amplification effect.(2)The effects of different sample pretreatment methods on the genomic DNA quantification of mutton were studied,including the repeatability and stability study of standard preparation methods,the comparative study of different extraction methods of animal genomic DNA,and the addition of different types and different contents in samples.The effect of matrix meat on the extraction of genomic DNA and qPCR amplification of mutton.The results show that the standard sample preparation method used in the experiment has good repeatability and stability.The magnetic bead method is more suitable for the extraction of animal genomic DNA in four extraction methods.The extraction and expansion of genomic DNA from different types of matrix meat There was almost no effect on the increase,and there was no significant difference in the experimental results.(3)Based on the gene copy number,the mathematical model of mutton content and Ct value in the sample was constructed.The qPCR quantitative detection method was further established.Finally,the sample was directly subjected to qPCR to calculate the mutton content in the sample.The experimental results show that the functional relationships established by the method have good linearity,and R~2 is greater than 0.99.The method has good repeatability and stability,and the detection limit is 5%,which has good accuracy.(4)Established ddPCR internal standard quantitative method,added DNA to the sample to extract DNA,and used double ddPCR to determine the copy number of target and internal standard,and construct the function of copy number ratio and sample quality of detection target and internal standard.The relationship shows that the function established by the method has a good linear relationship,R~2 is greater than 0.99,and the stability,repeatability and accuracy of the method meet the quantitative requirements.The quantitative detection limit is 5%,which can be applied to the actual detection.(5)Applying the qPCR absolute quantification method and ddPCR internal standard quantification method established in this paper,28 samples of mutton samples provided by Chengdu Customs Technology Center were quantitatively tested,and the detection results of two quantitative methods were analyzed.In summary,this study is based on qPCR and ddPCR to establish a quantitative detection method for sheep-derived components,which can quickly and effectively quantitatively detect the sheep-derived components in the sample,with strong specificity and accurate results,and can be used by relevant law enforcement agencies.Provide a reliable basis.