Study On Identification And Quantitative Detection Of Mulberry Source Ingredient In Fruit Juice

With the popularity of common fruit varieties in recent years,people are more and more fond of the third generation fruits such as mulberry,raspberry and cherry and so on.Because the third generations fruits have good taste,abundant nutrition and rich nutritional factors with health care functions,the price of fresh food or for the development of food or health products is quite expensive.As a result,its products often appear to be fake and shoddy.For example,some mulberry processing products do not contain mulberry,but use cheap fruits or flavoring to achieve the effect These not only deceive consumers,but also greatly disrupt market order.Using modern bioinformatics methods and digital PCR technology,a rapid and accurate qualitative and quantitative detection method can be established,which can provide monitoring technical means for ensuring food quality and safeguarding consumers’ interests.In this study,the bioinformatics method was used to download the genomes of a variety of common fruits using the perl language program,and the specific genes of mulberry were screened through BLAST sequence alignment.The target sequences were selected from 29,262 screened sequences for primer design.At the same time,real-time fluorescent PCR and digital PCR primers and probes were designed according to the sequence of mulberry.The specificity,concentration and optimum annealing temperature of primers and probes were screened and optimized.Qualitative and quantitative detection methods of mulberry specificity were established.The sensitivity,detection limit and applicability of the method were tested.(1)Establishment of regular PCR method for mulberry species specificity identification: Through verification of mulberry specificity,optimization of PCR reaction system and procedure conditions,and determination of sensitivity of the method,the PCR method was established.The results indicated that the method had good species specificity with the optimum annealing temperature of 56 °C,and the primer concentration at 0.6 ?mol/L for the optimized amplification conditions.The samples with 0.05% mass fraction(DNA,25 ng/?L)were detected,and the repeatability was high.This method could positively detect the mulberry components in the samples when it was used to detect pure mulberry juice,mixed mulberry juice with orange juice,mixed mulberry juice with peach juice and apple juice,mixed mulberry juice with pear juice.(2)Establishment of a quantitative real-time fluorescence PCR(qPCR)method for mulberry specificity identification: Through optimization of mulberry species qPCR reaction system,specificity and sensitivity detection,standard curve of mulberry species-specific genes was generated based on standard DNA solution amplification curve,and quantitative detection of mulberry components was completed.The results showed that the method had high specificity and sensitivity.The quantitative detection limit of mulberry genomic DNA was 0.006 ng/?L.The standard curve equation of mulberry DNA amplification was y = 3.529 x + 127.18,R=0.995,and the amplification efficiency was 92.032%.It could be used for quantitative detection of mulberry processed products.This method can be used to detect pure mulberry juice containing 0.01% mulberry ingredients and to identify the mixed mulberry juice.(3)Establishment of a digital PCR method for mulberry specific identification: On the basis of primers and probes screened by real-time fluorescent PCR,the optimum concentration of primers and probes,annealing temperature,quantitative linear range and LOQ verification were determined.The optimal concentration of primers and probes was 0.4 μmol/L,the optimum annealing temperature was 57.9 °C,the quantitative detection limit was 0.006 ng/μL of mulberries genomic DNA.The mulberries genomic DNA can be calculated according to the curve equation of digital PCR method,which can be used for quantitative detection of mulberry processed products.To sum up,based on bioinformatics,the species-specific molecular biological identification methods for mulberry were developed by combining the qualitative PCR,qPCR and digital PCR technology.This method is suitable for adulteration identification of mulberry juice and its processed products,and can provide technical support for qualitative and quantitative detection of mulberry derived ingredients in food.