中国农业科学 ›› 2008, Vol. 41 ›› Issue (1): 195-200 .doi: 10.3864/j.issn.0578-1752.2008.01.026

• 贮藏·保鲜·加工 • 上一篇    下一篇

PCR法快速检测扩张青霉

樊明涛,毕静莹,Mansel W Graffith,Wang Haifeng   

  1. 西北农林科技大学食品科学与工程学院
  • 收稿日期:2006-09-22 修回日期:1900-01-01 出版日期:2008-01-10 发布日期:2008-01-10
  • 通讯作者: Mansel W Graffith

Rapid detection of Penicillium expansum using PCR technology

  1. 西北农林科技大学食品科学与工程学院
  • Received:2006-09-22 Revised:1900-01-01 Online:2008-01-10 Published:2008-01-10

摘要: 【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5×10-6 μgDNA/每反应体系,整个检测时间约为5 h,比传统培养法(4~6 d)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。

关键词: 苹果, 扩张青霉, PCR, 快速检测

Abstract: Patulin is the major mycotoxin in apple juice produced in apple juice processing by penicillium expansum, rapid detection of penicillium expansum is of great significance for controlling the fungi and ensuring apple juice quality. Based on isoepoxydon dehydrogenase (primer ISO) and polygalacturonase (primer POL) respectively, two pairs of primers were used to amplify DNA from penicillium expansum and compare the specificity of penicillium expansum DNA amplification. It has been proven that primer pairs(ISO) have poor specificity, while primer pairs(POL) have high specificity and only 404 bp DNA fragments were amplified. It has also been shown from DNA serial dilution amplification sensitivity that the amplification sensitivity of DNA serial dilution from plate culture has at least one fold level higher than that from liquid culture. DNA concentration determination has also proven that DNA concentration from plate culture is much higher than that from liquid culture. The POL primers also had a high amplification specificity of DNA from penicillium expansum inoculated apples and juice, while there were difference of DNA amplification strongness from different apple species. Apple DNA had no interference with amplification of penicillium expansum DNA