九香虫药材及其混伪品的PCR鉴别

PCR Identification of Aspongopus chinensis and the Adulterants

目的:建立一种能够准确鉴别九香虫及混伪品的特异性PCR方法,解决形态相似动物药材的鉴定问题。方法:收集不同产地九香虫及其常见混伪品8种123份,基于九香虫及其混伪品的细胞色素C氧化酶亚基I(COI)序列差异,利用Clustal W软件进行同源比对并且筛选出特异性单核苷酸多态性(SNP)变异位点,根据变异位点设计特异性鉴别引物,采用三步法进行PCR扩增,并对影响PCR反应体系的反应条件进行考察,实测鉴定了九香虫及混伪品。结果:当模板量30~100 ng,退火温度为55℃,循环次数为35次时,九香虫能扩增出301 bp的单一明亮条带,而其混伪品均不具有此特异性条带。结论:使用特异性引物对九香虫及其混伪品进行验证的结果良好,与传统形态鉴定结果基本一致。因此,通过特异性PCR方法可以快速准确地鉴定正品九香虫。

Objective:To establish an efficient and specific polymerase chain reaction PCR method for the identification of the Aspongopus chinensis and its adulterants,so as to solve the identification problem of animal medicinal materials with similar morphology.Methods:A total of 123 samples from 8 species of Aspongopus chinensis and its adulterants were collected in different places.Based on the cytochrome C oxidase subunit I(COI)sequences,Clustal W software was used to conduct homology comparision and the specific single nucleotide polymorphism(SNP)loci were screened.Specific primers were designed according to the mutation loci,PCR amplification was carried out by 3-step method,and the reaction conditions affecting the PCR reaction system were investigated,the Aspongopus chinensis and its adulterants were identified.Results:When the template was 30 to 100 ng,annealing temperature was 55℃and cycles number was 35,a single bright band of 301 bp was amplified from Aspongopus chinensis.However,its adulterants did not have the specific bands.Conclusion:The results of using specific primers to verify the Aspongopus chinensis and its adulterants are good,the results were consistent with traditional morphological identification.Therefore,the specific PCR method can be used to quickly and accurately identify the genuine Aspongopus chinensis.