| With the improvement of people's living quality,people's pursuit of nutritional value is also getting higher and higher.Food safety and quality have become the focus of people's attention.Cherry,as one of the third generation fruit varieties,has very high nutritional value and functional ingredients.It has the functions of reducing diabetes,inhibiting metabolic disorders,antioxidant,anti-inflammatory,anti-cancer and so on.Because of the high value,delicious and unique taste,in order to gain more profits,it is possible to produce the adulterated cherry juice and other products.Therefore,rapid and accurate detection and identification of cherry components in juice can provide technical support for quality control of cherry and its products.(1)Finding the specific sequence of cherry species.A total of 41 genomes from 19species of common fruits,such as cherry,strawberry and kiwifruit,were obtained by NCBI.Sequence BLAST was carried out by Perl language.Finally,nine pairs of primers with high specific amplification effect were selected,their amplification products were sequences,and then the specific regions of cherry genome were determined.(2)Establishment of specific PCR method for cherry species identification.The genomic DNA of 19 species of common fruits,such as cherry,strawberry and kiwifruit,was used as template to detect the specificity of the primers,optimize the PCR system and annealing temperature,and determine the sensitivity of the primers.The results showed that among the 9 pairs of primers,primers che36-F/R had the best amplification effect and specificity.When the concentration of primer was 0.4?mol/L and annealing temperature was 58°C,the sensitivity of PCR reached 0.5%.The actual sample tests showed that this PCR method could be used for qualitative identification of cherry components in the mixed fresh juice and non-fresh juice.(3)Establishment of a specific real-time fluorescent PCR method for cherry species identification.Two pairs of primers and probes were designed according to che36 sequence which was identified as the specific region of cherry genomic DNA.After optimized the reaction system and program of qPCR,the primer concentration and the probe concentration of che36-1-QF/QR/QP were both 0.5?mol/L,and the sensitivity of qPCR would reached 0.1%of cherry genomic DNA.Under the optimal amplification conditions,the standard curve was y=-3.356x+31.062,R~2=0.998,and the amplification efficiency was98.545%.There was a relationship between the Ct value and the DNA template quantity of cherry genomic DNA with a correlation coefficient R~2=0.9985.Juice sample tests showed that this method could be used to qualitatively identify cherry components in the mixed fresh juice and non-concentrated juice,and to determine the content of cherry genomic DNA.(4)Establishment of a specific digital PCR method for cherry species identification.After optimized the reaction system and program of ddPCR,the primer concentration and the probe concentration of che36-1-QF/QR/QP was 0.5?mol/L and 0.2?mol/L,and the annealing temperature was 56.7°C,the ddPCR amplification results was the best.The detection limit under the linear quantitative range was 0.24ng of cherry genomic DNA.There was a linear relationship between the number of copies of che36 and the weight of DNA templates with a correlation coefficient R~2=1.Juice sample tests showed that this method could be used to qualitatively identify cherry components in the mixed fresh juice,non-concentrated juice and concentrated juice,and the number of che36 copies which was the specific DNA fragment of cherry could be obtained.In this study,a species specificity identification method for cherry was established,which provided technical support for identification of cherry-derived components in fruit juice and experimental basis for food quality and safety control. |
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