基于gpd基因的大蒜黑腐病菌实时荧光定量PCR鉴定技术  

Detection of Embellisia allii Using Real-Time Quantitative PCR Based on Glyceraldehyde-3-Phosphate Dehydrogenase Gene

作　　者：王英超[1] 甘琴华[1] 厉艳[1] 纪瑛[1] 吴兴海[1] 邵秀玲[1] 

机构地区：[1]山东出入境检验检疫局,山东青岛266002

出　　处：《中国农业科学》2015年第2期390-397,共8页Scientia Agricultura Sinica

基　　金：国家质检总局项目(2009IK254;2013IK173);山东出入境检验检疫局项目(SK201307)

摘　　要：【目的】大蒜黑腐病菌(Embellisia allii(Campanile)Simmons)的检疫鉴定主要采用形态鉴定方法。不仅耗时长,而且大蒜黑腐病菌与本属其他种以及其他属的分生孢子非常相似,极难分辨,需要建立快速、有效的分子诊断方法。研究旨在建立实时荧光定量PCR(real-time quantitative PCR,q PCR)体系以准确、灵敏地鉴定大蒜黑腐病菌。【方法】从NCBI的Gen Bank数据库中,选取大蒜黑腐病菌和其他大蒜病害以及近似种的3-磷酸甘油脱氢酶(gpd)基因核酸序列数据15种,应用引物设计软件Primer express,根据探针设计原则从理论上选出1条探针和3对引物。根据试验结果对上述引物进行筛选,筛选出能够鉴定出大蒜黑腐病菌的特异性引物对和Taq Man探针。同时对反应体系中复性-延伸温度的反应参数进行优化,设计56、58、60℃3个温度梯度;将大蒜黑腐病菌基因组DNA在紫外核酸分析仪上进行定量后进行10倍的梯度稀释,对探针灵敏性进行测试,从而建立大蒜黑腐病菌Taq Man水解探针法q PCR检测体系。【结果】从基于Gen Bank上的15种gpd基因核酸序列设计的引物和探针中,筛选出能够鉴定大蒜黑腐病菌的特异性引物对Emb21/Emb32和Taq Man探针Emb。在供试的6种菌株中,只有靶标菌大蒜黑腐病菌利用该引物对和探针能够被特异性的检测到阳性,空白对照和其他参试菌均没有荧光信号的增加,检测为阴性。复性-延伸温度反应参数优化中,△Rn荧光信号增加值在58℃时达最大,56、60℃荧光信号增加值降低,经过多次重复试验,确定最佳的复性-延伸温度为58℃。灵敏度测试中,大蒜黑腐病菌DNA浓度稀释到140 pg时,所得的图形较好;当体系中模板浓度稀释到14 pg时,所得的图形较差。最终DNA检测灵敏度为140 pg。应用该引物和探针建立的q PCR检测方法能够从供试的菌株中特异性地检出大蒜黑腐病菌。【结论】建立了大蒜黑腐病菌Taq M[Objective] Morphological identification method is mainly used for detection and identification ofEmbellisia allii （Campanile） Simmons. The method is time-consuming in identification, moreover, the spore of garlic bulb canker is very similar to other species of the genus Embellisia and other genera, but is also very difficult to distinguish. Rapid and effective molecular diagnostic methods are needed. The objective of this study is to develop a real-time quantitative PCR （qPCR） technique for detection of the E. allii sensitively and accurately. [Method] From the NCBI GenBank database, 15 kinds of glyceraldehyde-3-phosphate dehydrogenase （gpd） gene nucleic acid sequences of the garlic bulb canker and other similar species diseases were selected,according to primer design software Primer express, a probe and three pairs of primers were elected theoretically. Based on the experimental results of the above-mentioned primers, specific primers and TaqMan probes to E. allii were screened. While the reaction system renaturation-extension temperature of the reaction parameters were optimized, three temperature gradients of 56, 58, 60℃ were designed. The garlic bulb canker pathogen genomic DNA in the nucleic acid analyzer UV quantification was performed 10 times serial dilutions in order to test the probe sensitivity. The TaqMan qPCR detection system was established with optimized reaction conditions. [ Result ] The species-specific primer pair Emb21/Emb32 and probe Emb were designed based on gpd gene for E. allii, and E. allii could be detected specifically from the 6 tested strains with qPCR based on gpd gene, only the target fungi could be specifically detected positive, the blank control and other strains were detected negatively. About the optimization of refolding- extended temperature reaction parameters, ARn fluorescence signal at 58℃ was the maximum while the value of the fluorescence signal was lower at 56, 60℃. After repeated tests, the optimal annealing-extended temperature was determined as 5

关 键 词：大蒜黑腐病菌 3-磷酸甘油脱氢酶基因 实时荧光定量PCR 

分 类 号：S436.33[农业科学—农业昆虫与害虫防治]