Quantitative Detection Technology Of Beef Source Components By QPCR And DdPCR

In recent years,adulteration of meat products has occurred frequently.The main way of adulteration is to adulterate high-priced meat products with cheap meat raw materials,and do not indicate the ingredients in commodity labels,which seriously damages the legitimate rights and interests of consumers.The existing identification standards of animal-derived ingredients are all qualitative testing standards,which can only meet the simple identification needs of adulterated meat products.However,with the continuous improvement of the adulteration level of illegal traders,the qualitative testing standard can not be accurately quantified,but the existing standards objectively protect the counterfeiters.Therefore,it is necessary to establish a stable,accurate and effective method for quantitative detection of the quality percentage of animal-derived ingredients,so as to provide a convincing detection result for law enforcement and supervision departments,and to ensure a good inspection without missing inspection.Beef nutrition is rich,market sales are huge,adulteration often occurs.This study intends to cattle,sheep chromosome gene for bioinformatics analysis,screening with constant gene copy number,and the target gene sequence,using fluorescence quantitative PCR and PCR technique,gene copy number and function relation of meat quality,completed a preliminary mathematical model building,realize the accurate quantitative on beef,so as to solve difficult to distinguish between low concentration of unintentional cross contamination and intentionally illegally added this technical problem,provide the basis for administrative law enforcement supervision.Based on real-time fluorescence quantitative PCR technology and micro-drop digital PCR technology,this paper established a quantitative detection method for beef source components.The main research contents and results are as follows:1.Specific primers and probes were designed for the study on the chromosomal single-copy gene GH of cattle and sheep breeds for daily consumption.The specificity of the established method was good,the absolute sensitivity of qPCR reached 0.01 ng,and the actual sensitivity was 0.01%.The absolute sensitivity of ddPCR method reached 0.21 copies/L.2.With phenol chloroform method,solution precipitation(DNeasy?mericon?Food faced Kit),centrifugal column method(DNeasy?Blood and Tissue Kit Kit)and the method of magnetic beads(day animal Tissue method of extracting genome magnetic beads Kit)to extract genomic DNA,comparison of different types of Kit genomic DNA extraction animal tissues.The results showed that the magnetic bead method combined with automatic nucleic acid extraction instrument had the best DNA quality,the highest stability and the shortest extraction time.3.To explore the effects of different matrix meat on the amplification of target meat species,pork,mutton and duck were selected as the matrix meat respectively,and the Ct values of the target gene and the dorsal marker gene were compared.The difference was not significant(p<0.05).Beef samples were treated with common flavoring salts and food additives(soy protein isolate,carragenan,sodium benzoate)to explore the effects of ingredients and additives on the extraction of genomic DNA amplification,and the differences were significant(p>0.05).There was no significant difference in beef samples treated with sugar and vegetable oil(p<0.05).4.Fluorescence quantitative PCR technology and microdroplet digital PCR technology were respectively used to establish the precise quantitative detection method of beef source ingredients by using absolute quantitative method and adding reference substance quantitative method.The results showed that the mathematical relationship based on DNA content and gene copy number had a good linear relationship,and the R~2 was greater than 0.99.5.Quantitative detection of bovine origin components:the practicability of the proposed quantitative method was verified in this paper by using simulated mixed samples and 30 commercial beef samples.Results show that the established mathematical model based on gene copy number of quantitative stability,better accuracy,using qPCR establish beef source components quantitative methods,the method of simulation samples validation and its relative standard deviation RSD is greater than 1%of the mixed samples than that of 25%,5%mixed samples the relative standard deviation less than 25%RSD,target meat kind of greater than 5%quantitative result was accurate;The cow source quantitative method was established by ddPCR,and the relative standard deviation RSD of this method was less than25%for the verification of simulated samples,meeting the absolute quantitative standard,which is helpful for the technical detection of the regulatory authorities in the anti-counterfeiting law enforcement.To sum up,the quantitative detection methods of real-time fluorescence quantitative PCR and microdroplet digital PCR established in this paper based on gene copy number can realize the quantitative detection of bovine origin components,which can be used as a basis to distinguish intentional addition from unintentional pollution and provide a strong technical guarantee for law enforcement and supervision departments.