目的: 建立基于线粒体细胞色素C氧化酶亚基Ⅰ(COⅠ)基因的分子标记技术,对中成药中土鳖虫(地鳖、冀地鳖)及其伪品进行分子鉴定的方法。方法: 收集不同地区土鳖虫正品及其伪品,对所有样品进行DNA的提取,通过对土鳖虫及其伪品DNA条形码COⅠ基因进行对比分析,设计药用土鳖虫的鉴别引物,引物序列为DBI-F(5’-GGCGTTGGCACAGGTTGGACT-3’)和DBI-R(5’-ATTTGTTCAGGTTTTATGTTG-3’)。采用两步法进行特异性聚合酶链反应(PCR)扩增,并对PCR反应体系和反应程序影响因素进行考察,从而建立特异性PCR技术对宫瘤消胶囊等11个中成药中土鳖虫的真伪进行鉴别。结果: 建立了土鳖虫快速特异性PCR反应程序,引物体系只针对土鳖虫成分DNA进行扩增,与中成药中其他药味的DNA均无交叉扩增。将其应用于11个品种13批次样品的检测,以1%琼脂糖凝胶电泳检测PCR产物,含正品土鳖虫的批次在250 bp附近处有一清晰条带,而阴性样品无条带。结论: 通过特异性引物PCR扩增、电泳检测和克隆测序的方法,可以有效鉴别宫瘤消胶囊等11个中成药中的土鳖虫成分真伪。该方法具有快速、特异、灵敏的优点,为中成药中土鳖虫掺伪鉴定提供了分子生物学方法。
Objective: To establish a molecular method based on the molecular marker technology of mitochondrial cytochrome C oxidase subunit Ⅰ(COⅠ) gene to identify Eupolyphaga Steleophaga(Eupolyphaga sinensis Walker, Steleophaga planeyi Boleny) and its counterfeit in Chinese patent medicine. Methods: Eupolyphaga Steleophaga and its counterfeit products from different regions were collected and their DNA was extracted. By comparing and analyzing the DNA barcode COⅠ gene of Eupolyphaga Steleophaga and its counterfeit product, the primers of DBI-F (5’-GGCGTTGGCACAGGTTGGACT-3’) and DBI-R’(5’-ATTTGT- TCAGGTTTTATGTTG-3’) were designed. Two-step method was used to amplify specific polymerase chain reaction (PCR), and the influencing factors of PCR reaction system and reaction procedure were investigated, and then established the specific PCR technology of identifying the authenticity of Eupolyphaga Steleophaga in 11 Chinese patent medicines such as Gongliuxiao capsules. Results: A rapid and specific PCR reaction procedure for Eupolyphaga Steleophaga was established. The primer system was amplified only for DNA of Eupolyphaga Steleophaga with no cross amplification from DNA of other herbs in Chinese patent medicine. It was applied to 11 varieties of 13 batches of samples. The PCR products were detected by 1% agarose gel electrophoresis. The batches of genuine Eupolyphaga Steleophaga showed a clear band near 250 bp, while negative samples had no stripe. Conclusion: The method of specific primers PCR amplification, electrophoretic detection and cloning and sequencing can effectively identify the authenticity of Eupolyphaga Steleophaga products of 11 Chinese patent medicines such as Gongliuxiao capsules. The method is rapid, specific and sensitive. It can provide a molecular biological method for identification of Eupolyphaga Steleophaga adulteration in Chinese patent medicine.
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