化风丹药母中3种优势微生物的实时荧光定量PCR方法的建立  

Establishment of a Real-Time Fluorescence Quantitative PCR Method for Three Dominant Microorganisms in Huafeng Pellet Yaomu

作　　者：袁小凤 舒淇琳 曹国琼 徐剑 陈有丽 张永萍 Yuan Xiaofeng;Shu Qilin;Cao Guoqiong;Xu Jian;Chen Youli;Zhang Yongping(School of Pharmacy,Guizhou University of Chinese Medicine,Guiyang 550025,China;National Engineering Technology Research Center of Miao Medicine,Guiyang 550025,China;Guizhou Engineering Technology Research Center of Processing and Preparation Traditional Chinese Medicine,Guiyang 550025,China;Zunyi Liaoyuanhetang Pharmaceutical Co.,Ltd.,Zunyi 563000,China)

机构地区：[1]贵州中医药大学药学院,贵州贵阳550025 [2]国家苗药工程技术研究中心,贵州贵阳550025 [3]贵州中药炮制与制剂工程技术研究中心,贵州贵阳550025 [4]遵义廖元和堂药业有限公司,贵州遵义563000

出　　处：《亚太传统医药》2024年第6期28-32,共5页Asia-Pacific Traditional Medicine

基　　金：国家自然科学基金(82360774);贵州省基础研究项目(黔科合基础-ZK〔2021〕一般517);贵州省教育厅滚动支持省属高校科研平台团队项目(黔教技〔2022〕022号);贵州省科技计划项目(黔科合中引地〔2023〕006);贵州省高层次创新型人才项目(黔科合平台人才-GCC〔2023〕037)。

摘　　要：目的:建立化风丹药母中3种优势微生物米曲霉(Aspergillus oryzae)、汉逊德巴利酵母菌(Debaryomyces hansenii)、阿氏芽孢杆菌(Bacillus aryabhattai)快速、有效的实时荧光定量PCR检测方法。方法:以米曲霉、汉逊德巴利酵母菌、阿氏芽孢杆菌的重组质粒为标准质粒,设计特异性引物并进行特异性、灵敏性及重复性实验。结果:3种优势微生物的熔解曲线不在同一峰值。米曲霉、汉逊德巴利酵母菌、阿氏芽孢杆菌的荧光定量PCR最低检测限度分别为0.256、514、5.08 copies/μL。不同质量浓度的标准质粒变异系数(CV)均小于5%。结论:研究表明以荧光定量PCR技术建立的米曲霉、汉逊德巴利酵母菌、阿氏芽孢杆菌的检测方法具有重复性好、特异性强、灵敏度高等优点,可用于化风丹药母发酵过程中微生物的检测及定量。Objective:Establishment of a rapid and effective real-time fluorescence quantitative PCR method for the detection of 3 dominant microorganisms Aspergillus oryzae,Debaryomyces hansenii and Bacillus aryabhattai in Huafeng Pellet yaomu.Methods:The recombinant plasmids of Aspergillus oryzae,Debaryomyces hansenii and Bacillus aryabhattai were used as standard plasmids for the real-time fluorescence quantitative PCR method,and specific primers were designed and specificity,sensitivity and reproducibility experiments were performed.Results:The results showed that the melting curves of the 3 dominant microorganisms were not at the same peak,indicating good primer specificity.The lowest detection limits of fluorescent quantitative PCR for Aspergillus oryzae,Debaryomyces hansenii and Bacillus aryabhattai were 0.256 copies/μL,514 copies/μL and 5.08 copies/μL,respectively,which indicated that the method had high detection sensitivity.the coefficients of variation(CV) of the standard plasmids with different mass concentrations were less than 5%,indicating that the method was reproducible.Conclusion:It was shown that the method established by real-time fluorescence quantitative PCR for the detection of Aspergillus oryzae,Debaryomyces hansenii and Bacillus aryabhattai has the advantages of good reproducibility,strong specificity and high sensitivity,and can be used for the detection and quantification of microorganisms in the fermentation process of the Huafeng Pellet yaomu.

关 键 词：化风丹药母 米曲霉 汉逊德巴利酵母菌 阿氏芽孢杆菌 实时荧光定量PCR 

分 类 号：R284.1[医药卫生—中药学]