目的 对半夏曲中4种优势微生物建立快速、有效的荧光定量PCR检测方法。方法 以枯草芽孢杆菌Bacillus subtilis、宛氏拟青霉Paecilomyces variotii、丝衣霉菌Byssochlamys spectabilis、黑曲霉Aspergillus niger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 4种优势微生物的熔解曲线峰单一。枯草芽孢杆菌、宛氏拟青霉、丝衣霉菌、黑曲霉的最低检测限度分别为584、622、0.272、500拷贝/μL。不同质量浓度的标准质粒变异系数（CV）均小于5%。结论 建立的枯草芽孢杆菌、宛氏拟青霉、丝衣霉菌、黑曲霉的荧光定量PCR检测方法具有特异性强、灵敏度高、重复性好等优点，可用于发酵类中药中微生物的检测及定量。

Objective To establish a rapid and effective method for quantitative PCR detection of four dominant microorganisms in Rhizoma Pinelliae Fermentata. Methods The recombinant plasmids of Bacillus subtilis, Paecilomyces variotii, Byssochlamys spectabilis, and Aspergillus niger were used as standard plasmids. Design specific primers and perform specificity, sensitivity and reproducibility experiments. Results The melting curves of the four dominant microbes were single. The minimum detection limit for Bacillus subtilis, Paecilomyces variotii, Byssochlamys spectabilis, and Aspergillus niger were 584, 622, 0.272, and 500 copies/μL, respectively. The coefficient of variations (CV) of different concentrations of standard plasmid were less than 5%. Conclusion In this study, the quantitative detection method of Bacillus subtilis, Paecilomyces variotii, Byssochlamys spectabilis, and Aspergillus niger had the advantages of strong specificity, high sensitivity and good reproducibility, which could be used for the detection and quantification of microbes in fermented Chinese medicine.

国家中医药管理局2015年中医药行业科研专项（201507004-03）