| Background:The nail of Pangolins and the fish maw as a valuable Chinese medicine,with the standards of national living continuous improvement,and the demand is increasing,the price is rising rapidly.There are a lot of mixed falsify appeared on the market,so accurate identification it is very necessary to the nail of Pangolins and the fish maw.It is difficult to identificate its base source by traditional methods,while the DNA molecular technology has been widely recognized because of high accuracy and good reproducibility.So we identificated the nail of Pangolins and the fish maw by DNA barcode identification.Purpose:We extracted the total DNA from the pangolin and the processings through the improved DNA extraction method,to identificate the Pangolins by the microbarcodes we designed.The DNA barcoding technology is used to determine the origin of the species of fish maw in Guangzhou.Methods:We can’t extract the DNA of the pangolin by the normal DNA extraction method,in this paper,we eventually determine the DNA extraction method of pangolin nail,through a lot of DNA extraction method,including improve the sample pretreatment,extend the digestion time,with Cyt b gene amplification and sequencing,to examine the results.Sequenced the COI pangolin fragments and Cyt b fragments,in combination with other known sequence,comparison analysis the DNA barcode of 7 kinds of pangolin sequence,calculate the interspecific and intraspecific genetic distance,make the barcoding gap,and construct phylogenetic tree,to determine suitable for a piece of DNA barcode research.Designed a length of about 150 bp’s mini bar code in COI fragments,by PCR amplification and sequencing,in combination with other known sequence,constructing phylogenetic trees,to identificate the commercial pangolin nail.Using the COI primers we designed to PCR amplification and sequence for the 11 municipal sales of fish maw samples,building UPGMA system development tree,and the cluster analysis is carried out.The species identification engine of The Barcode of Life Data Systems has been compared to the existing fish sequences in the database,to identify the basis sources for each sample.Result:The total DNA from the pangolin and the processings were extracted by the improved DNA extraction method,amplified and sequenced by Cyt b,to prove the DNA is of the pangolin.It’s successfully amplified by COI sequences and Cyt b sequences,and the NJ tree were constructed shows pangolin and mix falsify can be differentiated.While the results show that based on COI sequences,the application of DNA barcode technology can be more effective to identify pangolin and mixed falsify.General COI fragments cannot to amplification the total DNA from the pangolin on market,while through the mini barcode can amplification not only the pangolin nails but also the processings.Through mini fragments to amplification 9 of the nails sold on market,the success rate of amplification and sequencing was 100%.By blast,8 of the nails were Manis Javanica and 1 of the nail was Manis pentadactyla.11 samples of fish maw can be amplified by the primer we designed,the success rate of amplification and sequencing was 100%.The results of BOLD identification showed that 11 samples were derived from 3 families of 5 species of fish,and the UPGMA cluster analysis confirmed the results.Conclusion:The total DNA from the and the processings were successfully extracted by the improved DNA extraction method,and the mini fragments we designed can be used to identify the pangolin in the market more effectively.The DNA barcode technology is combined with the BOLD identification system,which can be used for accurate basis source identification. |
PDF Full Text Request