小活络丸中小麦粉掺伪实时荧光PCR检测方法的建立  

The Establishment of Real-time PCR Detection Method for the Adulteration of Wheat Powder in Xiaohuoluo Wan

作　　者：任秀[1] 王菲菲[1] 李静[1] 白继超 张聿梅[1] 马双成[1] 崔生辉[1] Ren Xiu;Wang Feifei;Li Jing;Bai Jichao;Zhang Yumei;Ma Shuangcheng;Cui Shenghui(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：《中国药事》2022年第1期66-77,共12页Chinese Pharmaceutical Affairs

摘　　要：目的:通过实时荧光PCR-高分辨熔解曲线(HRM)联合分析技术,建立小活络丸(大蜜丸)中小麦粉掺伪快速、准确鉴别方法。方法:通过优化前处理方法,提取小活络丸样品的基因组;通过文献检索和生物信息学分析,得到小麦粉特异性引物;使用实时荧光PCR-HRM技术,对10批次小麦(粉)、16种其他禾本科植物及7种常见高淀粉食物样品进行特异性扩增,采用克隆测序对结果进行再确认;考察实时荧光PCR-HRM方法的检出限、精密度和重复性,同时对市售的小活络丸样品进行检测。结果:试验筛选获得了适用于本研究的特异性引物,建立了实时荧光PCR-HRM小活络丸掺伪小麦的检测方法;小活络丸样品掺伪小麦粉的检出限为0.01 g·g^(-1),试验精密度C_(t)值为26.63(RSD=2.15%),T_(m)值为89.53℃(RSD=0.05%);重复性Ct值为26.65(RSD=3.20%),T_(m)值为89.53℃(RSD=0.12%)。试验对市售的4个厂家9批次共计81份样品进行分析,确定了3批次来源于A厂家的小活络丸样品存在小麦粉掺伪的问题,将PCR扩增产物克隆测序,其结果为小麦(Triticum Aestivum L.)。结论:本研究建立了实时荧光PCR-HRM检测方法,可以快速判定小活络丸中小麦粉掺伪样品。Objective:To establish a fast and accurate identification method for adulteration of wheat powder in Xiaohuoluo Wan by real-time PCR combined with high-resolution melting curve(HRM)analysis technique.Methods:The genome of the sample of Xiaohuoluo Wan was extracted by optimizing the pretreatment method.The specific primers of wheat flour were obtained by literature retrieval and bioinformatic analysis.The samples(10 batches of wheat,16 batches of Gramineae plants and 7 batches of high-starch food)were amplified by real-time fluorescence PCR-HRM technique,and the results were further confirmed by clone sequencing.The limit of detection(LOD),precision and repetitiveness were investigated so as to validate the real-time fluorescence PCR-HRM method.In addition,the commercially available samples of Xiaohuoluo Wan were also tested.Results:Specific primers suitable for this study were screened,and a real-time fluorescence PCR-HRM detection method for adulteration of wheat powder in Xiaohuoluo Wan was established.The LOD of adulterated wheat flour in Xiaohuoluo Wan was 0.01 g·g^(-1).The precisions(RSD,%)were 2.15%for response curve(Ct value,26.63)and 0.05%for high-resolution melting curve(T_(m)value,89.53℃),respectively.And the repetitiveness values(RSD,%)were 3.20%for Ct value(26.65)and 0.12%for T_(m)value(89.53℃),respectively.A total of 81 samples of 9 batches of Xiaohuoluo Wan from 4 manufacturers on the market were analyzed in the test,and it was found that 3 batches of samples of Xiaohuoluo Wan from Manufacturer A were adulterated with wheat flour.The PCR amplification products were verified by cloning and sequencing assay,and the adulterated goods were wheat(Triticum Aestivum L.).Conclusion:A real-time fluorescence PCR-HRM detection method was estalbished to quickly identify the adulterated wheat flour in Xiaohuoluo Wan.

关 键 词：小活络丸 小麦粉 实时荧光PCR 高分辨率熔解曲线 禾本科植物 

分 类 号：R95[医药卫生—药学] R28