Development Of Reference Materials For Chicken And Duck DNA And Their Application To The Quantitation Of Meat Product Origin

The detection of animal-derived components is of great significance for food safety,import and export trade,halal food recognition,animal disease prevention and control and so on.The lack of reference materials seriously affects the reliability,traceability and mutual recognition of the test results.As the carrier of genetic information,nucleic acid has the characteristics of stable structure,long shelf life and good specificity,so it is an ideal material for the preparation of reference materials.In this paper,based on the highly conserved sequence of mitochondrial DNA,chicken-derived ND1 gene DNA reference materials and duck-derived cytb gene DNA reference materials were developed,and a quantitative detection method of chicken and duck origin components based on reference materials andPCR method was established to realize the quantitative detection of chicken and duck origin components.The results are as follows:1.The dry powder quality control samples were prepared by vacuum freeze drying,and a large amount of DNA was extracted by the self-improved CTAB method.The concentration,purity,specificity and cross contamination of the extracted products were verified.The results showed that the extracted products met the requirements of standard material candidates.2.The highly conserved sequences of chicken-derived and duck-derived genes were screened,and the sequences in the national standard GB/T 38164-2019"Real-time fluorescencePCR method for detection of common livestock and poultry animal origin"were selected to verify the sequence,establish and optimize the ddPCR reaction system,and determine the detection limit.The lowest quantitative limits of the established ddPCR detection method for chicken and duck origin are 4.60 copies/μL and 0.28 copies/μL respectively,and the linear correlation coefficients R~2 are more than 0.99.The reproducibility and reproducibility RSD of the method are less than 1.0%and 1.5%,respectively.3.The homogeneity and stability of reference material candidates were evaluated by optimized ddPCR method,and eight qualified laboratories were used to determine the value of reference material candidates.The characteristic values of chicken ND1 gene DNA reference material and duck cytb gene DNA reference material were determined to be6.62×10~7 copies/μL and 3.15×10~7 copies/μL,respectively,and the extended uncertainties were 2.88×10~6 copies/μL and 1.09×10~6 copies/μL,respectively.The two kinds of DNA reference materials prepared have good homogeneity and can be stored stably at-20℃for 6months.4.Based on the developed reference materials,the quantitative detection method of chicken and duck source components was established by ddPCR and qPCR methods,with the copy number concentration as the intermediate value.The absolute error was less than±10.0%.95 batches of market samples were tested,of which the detection rate of chicken-derived components was 22.1%and that of duck-derived samples was 21.1%.