关于中成药水蜜丸制剂掺伪米粉监管方法的研究

目的： 为了解决中成药水蜜丸掺伪米粉无检测方法的问题，引入普通PCR方法和荧光探针PCR 方法并制定相应新标准，以完善中成药掺伪检测的监管体系。方法：以通宣理肺丸为例，着重介绍引入PCR方法在完善中成药米粉掺伪检测体系中发挥的新作用。方法通过优化样品前处理和DNA提取过程，采用生物信息学分析和试验验证对检索得到的引物和探针特异性进行评估，并对2种方法的特异性、检出限、精密度和重复性进行考察和比较，对市售样品进行筛查。结果：试验通过评估DNA分子提取质量，选定了天根DNA提取试剂盒；建立了普通PCR方法和荧光探针PCR方法，并获得了适合于本研究的特异性引物和探针；2种PCR方法的检出限均为1 g·Kg^-1；普通PCR方法精密度和重复性良好，荧光探针PCR方法的精密度RSD为1.56%(Ct值=29.74)，重复性RSD为1.98%(Ct值=29.88)。 试验对3个厂家9批次样品进行筛选，结果表明6批次A厂家的样品掺入了米粉。PCR产物经过克隆测序后，与NCBI数据库比对，确认为水稻(Oryza sativa L.)。根据不同PCR检测方法的特点，通过参考其他行业标准，首次制定了中成药掺伪米粉的PCR检验检测标准。结论：建立了水蜜丸中普通PCR和荧光探针PCR掺伪检测方法并制定相应检测标准，通过新方法和新标准的建立，弥补了掺伪检测在监管中的漏洞，完善了中成药监管的标准化体系。

Research on the Supervision Method about the Detection of Adulterated Rice in Chinese Patent Medicines

Objective: In order to solve the problem that there is no detection method for the detection of adulterated rice in water pills, the conventional PCR and fluorescence probe PCR method were introduced and corresponding new standards were formulated to improve the supervision system for the adulteration detection in Chinese patent drugs. Methods: Taking Tongxuan Lifei Wan as an example, the new role of PCR in perfecting the adulteration detection system of Chinese patent medicine rice flour was introduced emphatically. The sample pretreatment and process of DNA extraction was optimized, the specificity of the retrieved primers and TaqMan probes was evaluated by bioinformatics analysis and experimental validation. The specificity, limit of detection(LOD), precision and repeatability of the two methods were investigated and compared, and commercial samples were screened. Results: TianGen DNA extraction kits were selected by evaluating the quality of DNA molecular extraction. The common PCR method and fluorescence probe PCR method were established, and the specific primers and probes suitable for this study were obtained. The detection limits were 0.001g in the two PCR methods. The conventional PCR method maintained good precision and reproducibility. In fluorescence probe PCR analysis, the precisions (RSD, %) were 1.56% (Ct =29.74) and the respectabilities were 1.98% (Ct = 29.88). Total 9 batches of samples from 3 factories were screened and the results show that 6 batches of samples from manufacturer A were adulterated with rice flour. The PCR product was compared with NCBI database by cloning and sequencing and confirmed as Rice (Oryza sativa L.). The PCR detection standards for adulterated rice flour of Proprietary Chinese medicine were established for the first time by considering characteristics of different PCR detection methods and referring to other industry standards. Conclusion: A conventional PCR and fluorescence probe PCR method was established to identify rice adulteration samples and corresponding detection standards were formulated. The new methods and standards were urgently needed for making up for the gap in the regulation of adulteration in Chinese patent medicines and enriching the supervision standardization system.