DNA Molecular Identification Of Testudinis Carapax ET Plastrum And Trionycis Carapax And Construction Of ACM Database

Testudinis Carapax ET Plastrum(TCP)and Trionycis Carapax(TC)are commonly used animal-derived medicines(ACMs)in TCM clinics.TCP is derived from the shell of Chinemys reevesii.It can nourish yin,suppress yang,strengthen kidney and bone,nourish blood and heart,secure menses and stop metrorrhagia.TC is derived from the shell of Pelodiscus sinensis.It can nourish yin,suppress yang,expel hectic fever and resolve hard lump.Due to the large demand,the same name of diverse origins,differences of traditional use,the similar morphological characteristics and lack of professional experience,some fake products of TCP and TC appeared on the market in recent years,which has a negative impact on the safety and effectiveness of medicines in clinical applications.Researchers have carried out some researches on the identification of TCP and TC and their original animals,but researches on the pulverized and the complex has not been studied deeply.Therefore,the scope of its application is limited.In this thesis,the primer sets are designed based on differences of mitochondrial genome sequences between CR and PS and their common fakes.And a specific,accurate and convenient technique is established for the identification of TCP and TC by specific PCR.It could provide a method to monitor the quality of market products and help to maintain the market system.Moreover,it is beneficial to the safety and effectiveness of clinical medication.Thus,it has important application value.Meanwhile,it could also provide a novel way for the identification of ACM.The main research contents are as follows:1.First of all,five specific primer sets are designed and screened based on differences of mitochondrial genome sequences between the CR and PS and their common fakes.Then,DNA is extracted and purified from TCP and TC by SDS-based protocol.The PCR reaction conditions are optimized by changing PCR reaction components and amplification program.And specificity and sensitivity are carried out under the optimum conditions.The results have indicated that the bands of DNA of TCP and TC and its common fakes(TS,MS and AF)are amplified at 120 bp,120 bp,81 bp,105 bp and 114 bp,respectively.What’s more,the cross reaction between primer sets were not observed,which showed strong specificity.It isindicated that the established technique based on species-specific primers could be used to identify the authenticity of TCP and TC.In addition,the limit of detection was estimated to be1 ng of genomes for all of five assays.And the corresponding species in adulterant are accurately detected.The research in this chapter laid the foundation for subsequent identification of raw materials and the pulverized of TCP and TC.2.Raw materials and processed products of TCP and TC are self-made.Whether the established technique based on species-specific primers could be used to these raw materials and processed products was investigated.In addition,the established method is used to identify commercial raw materials and processed products of TCP and TC,and judge the authenticity and adulteration according to the criterion.The results showed that self-made and commercial raw materials and processed products were amplified the same strip with the target fragments.And it is indicated that the technology not only successfully identified original samples and raw materials of TCP and TC,but also can be used to identify processed products of TCP and TC prepared by long time heating.3.Highly processed products of TCP and TC are self-made.Whether the established technique based on species-specific primers could be used to these highly processed products was investigated.And commercial highly processed products of TCP and TC are identified.Thereafter real-time PCR is used to further improve the detection sensitivity of identification technology.The results have indicated that real-time PCR identification technology could detect the corresponding species in all self-made highly processed products,and the detection effect is better than conventional PCR.However,the corresponding species in commercial highly processed products were not all detected.In addition,the effect of the DNA template in highly processed products extracted and purified by different extraction methods is compared.The results have indicated that three kit methods are beneficial to the preparation of DNA template with high amplification efficiency,in which animal genomic DNA extraction kit of Ezup column method is the best,and microgenomic DNA Extraction Kit of MagicMag Beads method is the next.4.Firstly,the framework of identification database of ACMs is designed.Then,Access is used to design columns and entry data.And Visual Studio is used to design the form.Moreover,ADODC control is added,and the codes are written into the code window of VisualStudio to connect the form to the Access target data.Finally,the retrieval function of the information of ACMs,specific primer sets and samples is realized through SQL statement.At last,the identification database of ACMs is initially constructed,which will help to further develop the network sharing platform to provide technical parameters and data support for identification researches of ACMs.And it provides convenience for the Authentication of ACMs.