Study On Identification Of Ranae Oviductus By Specific PCR Based On Whole Mitochondrial Genome

Ranae Oviductus is a precious traditional Chinese medicine(TCM),but there has been a controversy on the its original animal in academic circles..Meanwhile,the sails of Ranae Oviductus on the market are also chaotic due to the various kinds of faked medicine or adulterants.To solve the problems mentioned,we intend to establish a specific PCR method to distinguish Ranae Oviductus from various kinds of adulterants by analyzing the whole mitochondrial genome.To study the original animal of Ranae Oviductus,samples of Ranae Oviductus collected in Changbai Mountain have been analyzed.In addition,we also use real-time fluorescence quantitative PCR to quantify the percentage of Ranae Oviductus in related products.The main procedures of the study are as followed.1 The complete mitochondrial genome analysis of original animalsThe complete mitochondrial genome sequences of Rana chensinensis,Rana dybowskii,Rana huanrensis,Rana amurensis,Rana nigromaculata,Rana catesbeianna and Bufo gargarizans are obtained from GenBank database.MEGA v7.0 is used to construct NJ phylogenetic trees of mitochondrial whole genome sequences.The result suggests that R.chensinensis,R.dybowskii and R.huanrensis are evolutionally close species.Through analysis of the multi-sequence alignment of COI,Cytb,12 S rRNA and whole mitochondria by Clustal W motif in MEGA v7.0,we find that the variability of the intergenic sequence of ND5 and ND6 is significantly greater than the COI,Cytb and 12 S rRNA.Therefore,the intergenic sequence of ND5 and ND6 is suitable for identification of related species.2 Establishment of a specific PCR method for identification Ranae OviductusAccording to the intergenic sequence of ND5 and ND6 based on R.chensinensis,R.dybowskii and R.huanrensis,the forward primer(Large Forward Primer,LFP)and the reserve primer(Large Reserve Primer,LRP)are designed.DNA of originalanimals(R.chensinensis,R.dybowskii,R.huanrensis,R.amurensis,R.nigromaculata and B.gargarizans)and standard of Ranae Oviductus are extracted by modified SDS method,respectively.The primer named LFP/LRP is used for specific PCR amplification and agarose gel electrophoresis of above DNA.The LFP/LRP amplificates to obtain 856 bp,692bp,1047 bp and 596 bp products in R.chensinensis,R.-dybowskii,R.huanrensis and R.catesbeiana,respectively.Nineteen samples of Ranae Oviductuses in Changbai Mountain area are identified by the specific PCR established in this study.The result shows that all of the nineteen samples are R.-dybowskii.In order to further verify the accuracy of above identification results,the SFP/SRP(Small Forward Primer/Small Reserve Primer)is designed based on the intergenic sequence of ND5 and ND6 for R.dybowskii.The amplified fragment size is290 bp.And the primers is used to amplify nineteen samples of Ranae Oviductuses in Changbai Mountain area,and further confirmed that they are all from R.dybowskii.3 Establishment of quantitative analysis method for Ranae OviductusA specific primer named QFP /QRP(Quantitative forward primer/Quantitative reserve primer)is designed based on the intergenic sequence of ND5 and ND6 of Ranae Oviductus for real-time fluorescent quantitative PCR.A series of standard samples(0%,10%,20%,30%,40%,50%,60%,70%,80%,90%,100%)are prepared by mixing cyclodextrin and Ranae Oviductus at different mass ratios.And DNA is extracted by modified SDS method,the standard curve of mass percentage ratio and Ct value is established by real-time fluorescence quantitative PCR.The standard curve is y=-11.649x+30.967,and the linear coefficient is 0.9809,which indicate that the established standard curve of Ranae Oviductus has good linearity at the range of mass percentage ratio 0%-100%.The innovation of this research lies in:1.The specific PCR technology is used to establish the Ranae Oviductus identification method based on the intergenic sequence of mitochondrial genome for the first time.2.The real-time fluorescence quantitative PCR is used for the quantitative analysis of Ranae Oviductus for the first time.The specific PCR established in this study can accurately identify R.-chensinensis,R.dybowskii and a variety of related species,and it is not necessary to sequence.Therefore,it can reduce the cost and apply widely.The quantitative analysis method of Ranae Oviductus established in this study can be used to determine the content of Ranae Oviductus and its related products to make up for the deficiencies of existing methods for determination of Ranae Oviductus.