The Study On Identification Of Pheretima By Highly Specific PCR

Objective:Establish a more economical,convenient and accurate method of identifying Pheretima aspergillum with highly specific PCR to solve the problem that medicine pheretima is difficult to accurately identify because of the complicated sources.Meanwhile,provide an efficient and accurate method of identifying Pheretima aspergillum for medicine quality control and further quality standard formulating in order to ensure drug safety and efficacy of medicines for patients.Method:1.Common earthworm species were collected in Shanghai,Guangzhou and other places,prepared a series of concentrations of alcohol and made specimens by anesthesia,fixing and bleaching in order to the follow-up extraction of genomic DNA and long-term preservation.With the help of the annelid taxonomy experts of ShangHai JiaoTong University Qiu Jiangping researcher and his doctor Jiang Jibao and literature materials,we used classical zoological taxonomy to clarify species of the earthworm samples2.After designing universal primers of 16SrRNA,12SrRNA and COI gene sequences according to related gene sequences in GenBank database and optimized PCR conditions,and COI,16SrRNA and 12SrRNA gene of 10 species of earthworms including Pheretima aspergillum were amplificated by PCR.PCR products were purified and sequenced at biotechnology company BGI.The sequenceing were proofreaded and spliced using DNAStar and CondonCode Aligner respectively.Then a similarity search was done by BLAST to validate if the COI,16SrRNA and 12SrRNA genes we get were accurate and reliable.3.The sequences of 16SrRNA,12SrRNA and COI were aligned using DNAMan soft ware for analysis their differences.The sequence that had more variatio n sites was selected to design some pairs of specific primers which can identify Pheretima aspergillum specifically.One of which was choosed to e stablish a method of identifying Pheretima aspergillum by highly specific PCR.In addtion,stability of the method and its influence of individual differences were studied.4.The universality and applicability of the primers with high specificity were detected,by using the established method to identify the Pheretima aspergillum of Cheinese medicine and its mixed powder.Result:1.After identification,ten species of earthworms including Drawida japonica japonica(Michaelsen,1892)and Amynthas aspergillum(Perrier,1872)etc.were collected.2.The DNA sequence of COI?16SrRNA and 12SrRNA gene were respectively 700bp?350bp and 320bp.The BLAST GenBank similarity search showed that COI?16SrRNA and 12SrRNA gene fragments were accurate and reliable.3.Multiple alignments and analysis by DNAMan indicated COI,16SrRNA and 12SrRNA genetic differences dot was 17.08%,15.84%and 45.43%.Select to Three pairs of specific primers were designed using 12SrRNA which has more different sites.The primers 12st/12stf were found with high specificity and were used to establish a method of highly specific PCR of identification of Pheretima aspergillum.The method has a good stability and will not be affected by individual differences.4.The method which was applied to the identification of commercially pheretima medicine and mixed powders had high accuracy and stability.Conclusion:The method of identification pheretima medicine by highly specific PCR has accurately identified pheretima original animal,commercial and mixed powder successfully,which breaks through the limitations of traditional classical identification methods.It also has an advantages of operations easy,convenient,reliable,stable isn' t affected by individual differences(different origin and different populations)and the experimental environment,showed.