平菇细菌性褐斑病病原菌RT-PCR检测方法的建立及其应用  

Development and Application of Real-time Fluorescent Quantitative PCR for Detection of Pseudomonas tolaasii

作　　者：徐岩岩[1] 陈璐[1] 李金萍[1] 谢学文[1] 石延霞[1] 李宝聚[1] 

机构地区：[1]中国农业科学院蔬菜花卉研究所,北京100081

出　　处：《园艺学报》2013年第1期169-178,共10页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(31071838)

摘　　要：平菇细菌性褐斑病是一种严重危害平菇生产的病害,早期监测和防治是关键。采用平菇细菌性褐斑病病原菌托拉斯假单胞杆菌(Pseudomonas tolaasii)毒素基因的特异性引物(Pt-1A)/(Pt-1D1),通过扩增条件优化,建立了该菌的实时荧光定量 PCR(Real-time fluorescence quantitative Polymerase ChainReaction)检测及富集方法,并利用该方法完成了该菌在平菇表层的动态监测。试验结果表明,实时荧光定量 PCR 对托拉斯假单胞杆菌的检测范围为 102～ 109cfu · mL-1,在经过选择性培养基富集后,检测灵敏度进一步提高了 100 倍。利用选择性培养基富集及荧光定量 PCR 检测方法,可在病害症状未显现之前检测到病原菌,为平菇细菌性褐斑病的流行监测和早期防治奠定了技术支持。Brown blotch disease caused by Pseudomonas tolaasii is an important mushroom disease. It can cause serious yield and quality loss. Early detection and monitoring of the pathogen is crucial step for disease control. A real-time fluorescent quantitative PCR by using specificity primer（Pt-1A）/（Pt-1D1）and enrichment method was developed to detect the bacterial pathogens of P. tolaasii. The method was uesd to analyze the bacterial population dynamic change of P. tolaasii on the Pleurotus ostreatus sporocarp surface. Our results showed that the detection range of this was from 102 to 109 cfu ？ mL-1,and the sensitivity with enrichment step can reach about 100-fold higher than that without enrichment step. We conclude that the real-time quantitative PCR with the enrichment method can be used to detect P. tolaasii at the early stage of infection,and can provide technical support for the mushroom bacterial epidemic surveillance and early prevention of the disease.

关 键 词：平菇 托拉斯假单胞杆菌 实时荧光定量PCR 

分 类 号：S646.14[农业科学—蔬菜学]