平贝母PCR-核酸试纸条快速检测方法的建立和评价  

Establishment and evaluation of a rapid PCR-colloidal gold test strip method for the detection of Fritillaria ussuriensis

作　　者：马玉贺 商聪慧 马秋贺 李涛 刘悦 潘蓓珍 高丽君 李明成 夏薇 曲永梅 MA Yu-he;SHANG Cong-hui;MA Qiu-he;LI Tao;LIU Yue;PAN Bei-zhen;GAO Li-jun;LI Ming-cheng;XIA Wei;QU Yong-mei(School of Medical Technology,Beihua University,Jilin 132013,China;Innovation Center for Detection on DNA Fingerprint of Traditional Chinese Medicine,Jilin 132013,China;Jilin Guoan Pharmaceutical Co.,Ltd.,Jilin 132013,China)

机构地区：[1]北华大学医学技术学院,吉林吉林132013 [2]吉林省中药DNA指纹检测技术科技创新中心,吉林吉林132013 [3]吉林国安药业有限公司,吉林吉林132013

出　　处：《药学学报》2024年第6期1773-1778,共6页Acta Pharmaceutica Sinica

基　　金：吉林省医药健康产业发展专项资助(20230401096YY);北华大学研究生创新计划项目资助(研创合字[2022]057号);吉林省大学生创新创业训练计划项目(202310201140);吉林省科技厅重点科技成果转化项目资助(20170307001YY).

摘　　要：本研究针对平贝母的ITS2序列设计特异性鉴别引物,优化反应体系及条件,构建PCR-核酸试纸条实现可视化检测平贝母。通过分子克隆及测序技术,构建平贝母DNA阳性对照品,制定质量标准。对建立的方法进行性能评价,检测其灵敏度、特异性和重复性,对市售样品进行真伪鉴定。结果显示,基于ITS2序列可对平贝母及其混伪品进行区分。正品平贝母PCR产物滴定于试纸条上显示T、C线两条带,伪品与阴性对照只显示C线一条带,与琼脂糖凝胶电泳结果吻合。特异性为100%,PCR-核酸试纸条灵敏度可达0.1 ng·μL^(-1),高于凝胶电泳检测10倍。16个平贝母市售样品中11个合格,5个不合格。综上,本研究建立的平贝母PCR-核酸试纸条检测方法特异、快速、精准、可视化,可为平贝母检测提供新的技术思路。This study design of specific identification primers for the ITS2 sequence of F.ussuriensis.The reaction system and conditions were optimized,and PCR-nucleic acid test strips were constructed to realize the visual detection of F.ussuriensis Bark.Through molecular cloning and sequencing technology,we constructed a positive control for F.ussuriensis DNA and formulated quality standards.The established method was evaluated for sensitivity,specificity and reproducibility,and the authenticity of the commercially available samples was identified.Results demonstrated that based on the ITS2 sequences,F.ussuriensis and its mixed forgeries could be distinguished.The PCR products of the authentic F.ussuriensis on test strips showed two bands in the T and C lines,while the pseudo products and negative control showed only one band in the C line,which was consistent with the results of agarose gel electrophoresis.The specificity was 100%,and the sensitivity of the PCR-nucleic acid test strip was up to 0.1 ng·μL^(-1),which was 10 times higher than that of the gel electrophoresis assay.11 out of 16 commercially available samples of F.ussuriensis were qualified,and 5 were unqualified.Collectively,the PCR-nucleic acid test strip method established in this study is specific,rapid,accurate and visualized,which can provide a new technical idea for the detection of F.ussuriensis.

关 键 词：平贝母 分子克隆 ITS2序列 核酸试纸条 可视化 

分 类 号：R917[医药卫生—药物分析学]