PCR法快速检测扩张青霉

Rapid Detection of Penicillium expansum Using PCR Technology

【目的】研究棒曲霉素扩张青霉菌(Penicillium expansum)的快速和特异性PCR检测方法。【方法】通过P.expansum的纯菌平板和液体培养以及纯菌接种于苹果、苹果果肉和苹果汁,用Cenis改良方法提取DNA,以基于isoepoxydon dehydrogenase和polygatacturonase编码基因的两对引物扩增P.expansum DNA,建立特异和快速的检测P.expansum的方法。【结果】基于polygatacturonase基因的引物具有很强的特异性,只有目标DNA片段被扩增,而基于isoepoxydon dehydrogenase基因的引物扩增特异性较差。纯培养可以检测到5×10-6μgDNA/每反应体系,整个检测时间约为5h,比传统培养法(4～6d)时间大大缩短。苹果、苹果果肉和果汁接种纯P.expansum后所提DNA都得到了很好的扩增,具有很高的特异性,不受苹果样品DNA的干扰。【结论】本研究建立的P.expansum的PCR检测方法特异、快速、灵敏,可用于商品果汁和苹果汁工业化生产中P.expansum的快速检测和质量控制,也可用于工艺过程对P.expansum污染的快速溯源。

[Objective] To study the fast and specific detective method of patulin-producing Penicillium expansum. [Method] Through pure culture of P expansum in malt extract agar and malt extract broth, and cultivating Penicillium expansum in apples, apple flesh and apple juice, the P. expansum DNA was extracted by using modified Cenis method. Two pairs of primers based on isoepoxydon dehydrogenase and polygalacturonase were used to amplify P expansum DNA, and a method to specifically and quickly detect P expansum DNA was established. [Result] Primers based on polygatacturonase have high specificity that only target DNA was amplified, however, primers based on isoepoxydon dehydrogenase have poor specificity. 5 ×10^-6μg DNA per reaction in pure culture could be detected. The detection time （about 5 h） was remarkably shortened compared with conventional plate culture method （about 4-6 d）. DNA from apples, apple flesh and apple juice cultured with P expansum was also remarkably amplified with high specificity, and without interference from apple DNA. [Conclusion] The PCR method established in this study is specific, fast and sensitive to detect Penicillium expansum, it can be used in detection of P. expansum in apple juice production and quality control, it can also be used for tracing P. expansum contamination in processing.