荧光定量PCR检测霉变烟叶中米曲霉方法的建立

Establishment of a Real-time PCR Assay for Detection of Mold Fungi in Tobacco

摘要:

为建立快速高效的烟草霉变微生物定量与定性的检测方法，本研究针对陈旧烟叶中分离的米曲霉的rDNA ITS1基因片设计一对特异引物，制备含ITS1基因的重组质粒作为阳性对照，建立了米曲霉基因组的SYBR Green I应该定量PCR检测方法。结果显示，标准曲线的相关系数为0.998，最低可检测浓度为101 copy/μL的阳性质粒，与其他的微生物的基因组不发生交叉反应，该技术可为检测烟叶霉变微生物提供快速可靠的检测手段。

Abstract:

To establish a rapid and effective detection method for mold fungi in tobacco, a real-time PCR assay based on SYBR Green I was developed with a pair of primers designed according to the rDNA ITS1 gene of Aspergillus oryzae isolated from commercial tobacco. A recombinant T-vector containing the ITS1 gene was constructed as a positive control. The correlation coefficient of the standard curve was 0.998 and was highly sensitive with a detection limit of 101 copy/μL of positive recombinant plasmid. The technology was specific toward the genome of Aspergillus oryzae without any cross-reaction with other microbe genome. The developed real-time PCR method in this study can be used for detection of mold fungi in tobacco.