九味羌活丸和香砂养胃丸制剂米粉掺伪检测方法的建立和质量标准的制定  

Establishment of quality standards and test method of rice adulteration in Jiuweiqianghuo Wan and Xiangshayanwei Wan

作　　者：王菲菲[1] 任秀[1] 李静[1] 白继超 张聿梅[1] 郑健[1] 崔生辉[1] 马双成[1] WANG Feifei;REN Xiu;LI Jing;BAI Jichao;ZHANG Yumei;ZHENG Jian;CUI Shenghui;MA Shuangcheng(National Institutes for Food and Drug Control,Beijing 100050,China)

机构地区：[1]中国食品药品检定研究院,北京100050

出　　处：《暨南大学学报（自然科学与医学版）》2022年第1期97-105,共9页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：科技部“食品安全关键技术研发”重点专项项目(2017YFC1601400)。

摘　　要：目的:建立荧光探针PCR检测方法并拟定质量标准,旨在实现快速、准确地检出九味羌活丸(水丸)和香砂养胃丸(水丸)中的掺伪米粉。方法:通过优化样品提取方法,有效提取九味羌活丸和香砂养胃丸复方的基因组;通过文献检索和生物信息学分析,筛选得到适用于本试验的米粉特异性引物和TaqMan探针,并优化PCR检测方法。实验对方法的特异性、检出限、精密度和重复性进行考察,对市售样品进行筛查,并拟定了中成药荧光探针PCR检测方法的检测标准。结果:本实验优化了样品的前处理方法,通过文献检索和生物信息学分析检索到3组特异性引物和探针,并筛选得到了适合本研究的引物和探针。荧光探针PCR方法的检出限为每克样品可检出0.001 g米粉。九味羌活丸精密度的C_(t)值为30.34,相对标准偏差(RSD)为1.23%;重复性的C_(t)值为29.88,RSD为1.98%。香砂养胃丸精密度的C_(t)值为35.43,RSD为2.63%;重复性的C_(t)值为35.09,RSD为2.11%。对市售的7个厂家13批次样品进行分析,检出来自A厂家的5批次九味羌活丸和2批次香砂养胃丸掺入了米粉。利用克隆测序的方法,对PCR产物进行再确认,经NCBI数据库比对,结果均为水稻(Oryza sativa L.)。本研究拟定了九味羌活丸(水丸)和香砂养胃丸(水丸)荧光探针PCR检测方法标准。结论:建立了米粉的荧光探针PCR检测方法,并拟定质量标准,可快速准确地鉴别出米粉掺伪的九味羌活丸和香砂养胃丸样品,为中成药荧光探针PCR方法的标准制订提供参考。Objective:To establish a fast and accurate method to detect rice powder in Jiuwei Qianghuo Wan and Xiangsha Yangwei Wan by a Taq Man-based real-time PCR.Methods:The pretreatment process of DNA extraction was optimized and rice-specific primers and TaqMan probes were searched from retrieval papers by bioinformatic techniques.The samples(10 batches of rice,16 batches of Gramineae plants)were screened with real-time PCR analysis to identify the probes specificity.The sequencing-based results were validated by using clone sequencing.The limit of detection(LOD),precision,and repetitiveness were considered to validate the real-time PCR method.In additional,the obtained Jiuwei Qianghuo Wan and Xiangsha Yangwei Wan samples were also tested.A Real-time PCR method for the detection of rice adulteration were considered by referring to existing standards.Results:Specific probes were identified in present study.The LOD was 0.001 g rice flour per gram samples.The precisions(RSD,%)were 1.23%for Jiuwei Qianghuo Wan(C_(t) value,30.34)and 2.63%for Xiangsha Yangwei Wan(C_(t) value,35.43),respectively.And the respectabilities(RSD,%)were 1.98%(C_(t) value,29.88)and 2.11%(C_(t) value,35.09),respectively.Totally 5 batches of Jiuwei Qianghuo Wan and 2 batches of Xiangsha Yangwei Wan from a same factory were detected.The results were verified by cloning and sequencing assay.And the Real-time PCR method for the detection of rice adulteration were established.Conclusion:A TaqMan PCR method was estalbished to identify rice adulteration samples.It could be a good example for adulteration tests for Chinese patent drug by using Real-time PCR method.

关 键 词：九味羌活丸 香砂养胃丸 米粉 荧光探针PCR方法 克隆测序 

分 类 号：R917[医药卫生—药物分析学]