Authentic Identification Technique Of Berry Or Berry Products On DNA Barcoding

As a high value-added fruit,berries are increasingly popular with consumers in daily life.But,shorter harvest and higher prices lead to more and more adulteration in berry and berry products.Nowadays,the authenticic identification of berries and berry based products is still in the initial stages in our country.Regarding this,our study used Sanger sequence to build an authenticity identification method based on DNA barcoding which is emerging recently,and applied to the authentication of berry products.The detection range relates to completely blended juice,products which is replaced by cheap and the detection of mislabeling.In the study of extraction method of genomic DNA from berries,six kinds of commercial plant extraction kits were employed to be compared and analyzed,and the PCR enzyme was determined in the PCR system.Results showed that Macherey-Nagel,TIANGEN and Magen kits obtained poor-quality DNA with severe degradation and pollution.Qiagen,Promega and Biotecon kits all could extract DNA,whereas DNA integrity and amplification efficiency of Biotecon and Promega kits were low to compare with Qiagen kit.Therefore,Qiagen kit can be used as an universal method of DNA extraction of berries in molecular detection.What's more,1-5TM 2×Master Mix High-Fidelity showed the highest amplification efficiency in DNA amplification.Through the amplification efficiency,sequencing success rate,sresolution of species by single loci and paired loci,the DNA barcode sequence of berries with easy standardization and high reliability was selected.Results showed that,short rbcL loci(250 bp),psbA-trnH loci(450 bp)and long matK loci(890 bp)had the best performance in authentication of berry.The highest success rate was found in rbcL,psbA-trnH and matK gene,they were all 100%.As the resolution of single loci,psbA-trnH?matK>trnLc/d>rbcL>ITS BEL1/3>ITS BEL2/3>trnLg/h in the compasrison of NCBI datebase.When combined,matK+psbA-trnH,rbcL+psbA-trnH and rbcL+trnLc/d can improve the resolution of species.Because of low amplification efficiency of trnLc/d compared to other three locis,rbcL,psbA-trnH and matK were finally choosed as the berry DNA barcoding sequence.What's more,the construction of phylogenetic tree has verified the results of the three locis in NCBI database.In addition,we have established a Sanger sequencing process in single samples and mixed samples through the effect of DNA damage and detection results by simulating pasteurization and high-temperature short-time sterilization.Results showed that,short sequence of rbcL were still amplified while psbA-trnH and matK degraded severely after high-temperature short-time sterilization treatment.Because of less effect under extremeconditions,rbcL can be used for all of the berry products,the application range of psbA-trnH and matK is less than rbcL,In the results of cloning,10%of blueberry,cranberry or mulberry can be detected in freshly-mixed juice and juice treated by LTLT or HTST.Finally,the Sanger sequencing process was successfully established,in which,single samples are sequenced directly and mixed samples are cloned and then sequenced.The identification method established above was adopted to commercially available samples of berries.Dried berries,berry jam,berry juice and berry juice beverage collected from market,a total of 33 samples(5 kinds of dried fruit,12 kinds of jam,16 kinds of fruit juice and juice beverage),were identified based on rbcL and psbA-trnH locis.With 33 samples,48.49%of components detected by DNA barcoding were consistent with the label of products,mislabeling accounted for 45.45%and 6.06%were failed.Results showed that,DNA barcoding technique can be used for the authentification of single components and complex components in berry products,and it provides new control and detection tools for the quality control of production and the monitor of import and export inspection and quarantine.