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| 关于中成药水蜜丸制剂掺伪米粉监管方法的研究 |
| Research on the supervision method about the detection of adulterated rice in Chinese patent medicines |
| 投稿时间：2021-09-19 修订日期：2021-11-16 |
| DOI： |
| 中文关键词: 中成药标准化体系 监管方法 米粉掺伪 普通PCR反应 荧光探针PCR方法 |
| 英文关键词: Standard system in Chinese patent drug supervision method rice adulteration a conventional PCR method a TaqMan based real-time PCR method |
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| 中文摘要: |
| 目的：为了解决中成药水蜜丸掺伪米粉无检测方法的问题,本文引入了普通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了新的作用。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到的引物和探针特异性进行评估,并对两种方法的特异性、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和荧光探针PCR检测方法,并获得了适合于本研究的特异性引物和探针；两种PCR方法的检出限均为1g/kg；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.)。本文根据不同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：本研究建立了水蜜丸中普通PCR和荧光探针PCR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。 |
| 英文摘要: |
| Objective: In order to solve the problem about no detection method for the detection of adulterated rice in water pills, the conventional PCR and the TaqMan based real-time PCR methods were established to improve the standardized system for the adulteration detection in Chinese patent drug. Methods: Tongxuan Lifei Wan was selected to demonstrate the innovative effects of PCR in the detection of adulterated rice in Chinese patent medicine. The process of DNA extraction was optimized and rice-specific primers and TaqMan probes were searched from retrieval papers by bioinformatics analysis and experimental validation. The limit of detection (LOD), precision, and repetitiveness were considered to validate the two PCR methods. And the collected Tongxuan Lifei Wan samples were tested based on the developed methods. Results: TianGen DNA extraction kits were selected based on the DNA molecule quality data. Specific primers and probes were also identified in the optimized conventional PCR and TaqMan based real-time PCR methods. The LODs were 0.001g rice flour per gram samples in the two PCR methods. The conventional PCR method maintained good precision and reproducibility. In real-time PCR analysis, the precisions (RSD, %) were 1.56% (Ct = 29.74) and the respectabilities were 1.98 % (Ct= 29.88), respectively. Total 9 batches of samples from 3 factories were tested and the positive results were obtained in 6 batches from A factory. The results (Oryza sativa L.) were verified byScloning and sequencing assay. The PCR methods and standards were established for the first time by considering methods characteristics and technical guides from other areas. Conclusion: A conventional PCR and a TaqMan PCR method were established to identify rice adulteration samples. The new methods and standards were urgent need to make up for the gap in the regulation of adulteration in Chinese patent medicines and enrich the supervision standardization system. |
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