黄瓜细菌性角斑病PMA-qPCR检测方法的建立和应用  

The Establishment of PMA-qPCR for Detecting Bacterial Angular Leaf Spot on Cucumber and Its Preliminary Application

作　　者：孔维文[1] 李云龙 王敬琦[1] 刘婷婷[1] 陈南[1] 金一[1] 何晓青[1] 

机构地区：[1]北京林业大学生物科学与技术学院,北京100083 [2]北京市植物保护站,北京100029

出　　处：《生物技术通报》2016年第2期70-75,共6页Biotechnology Bulletin

基　　金：国家自然科学基金青年科学基金项目(51108029);中央高校基本科研业务费专项资金资助项目(TD2012-03);北京市优秀人才培养资助项目(2013D002006000001)

摘　　要：黄瓜细菌性角斑病为黄瓜常见的一种细菌病害,其致病菌为丁香假单胞菌黄瓜致病型。目前该病在全球范围内已经造成严重的经济损失。针对于PMA染料能够区分细胞死活的特性将其与荧光定量PCR技术结合。(1)通过比对分析Gen Bank中该菌种不同株的gap1基因序列,找出gap1基因的保守区并根据保守区基因序列设计了一对特异性引物Dxf1和Dxr1,以其他5种非丁香假单胞菌基因组为模板进行实时定量PCR扩增,只有丁香假单胞菌有扩增曲线,证明引物非常特异。(2)以gap1基因为目的基因构建其克隆载体,将克隆载体导入到大肠杆菌感受态细胞中进行培养,使其大量复制。再将质粒提取,以提取的质粒作为标准品,根据其浓度将其稀释为5×101-5×107 7个梯度绘制标准曲线,获得的扩增效率为96.6%,并做组内重复和组间重复,变异系数均在2%内,证明标准曲线重复性良好。(3)将构建好的方法用于实际样品的检测,得到的Ct值为27.99,根据标准曲线所得的起始模板与Ct值之间的线性关系公式,检测到100 mg患病叶片中含有的菌体为7.69×102拷贝。研究表明,该方法可以快速鉴定并检测实际样品中的活的致病菌的数量,为黄瓜细菌性角斑病的防控提供技术支持。Bacterial angular leaf spot on cucumber is a common bacterial disease for cucumber, and its causative pathogen is Pseudomonas syringae pv. lachrymans. Currently this disease has caused a worldwide serious economic loss. Duo to the feature of PMA dyes that may distinguish whether the bacterial cells are alive or dead, PMA-q PCR was established by combining it with fluorescence quantitative PCR（q PCR）technology.（1）We compared the sequences of gene gap1 in different strains of the bacteria in Gen Bank, and found out the conservative area of gene gap1, and designed a pair of specific primers Dxf1 and Dxr1 for this bacterium according to the sequences of conservative area of gene gap1. Then we applied the q PCR method to do amplification using the genome of 5 non-Pseudomonas syringae pv. Lachrymans as template, and only P. syringae pv. lachrymans showed the positive result, proving that primers had specificity.（2）We used the gene gap1 as the target gene to construct a clone vector that was inserted into the competent cells of Escherichia coli. Then the E. coli cells were cultured to have a lot of copies. Using the extracted plasmids as the standard sample, and it was diluted to seven gradients of 5×101-5×107 and then drew a standard curve. The amplification efficiency obtained from the standard curve was 96.6%, and the coefficients of variation both within groups and between groups were less than 2%, showing a fine repeatability of the standard curve.（3）We applied the PMA-q PCR method to detect the actual samples and gained a Ct value of 27.99. Based the linear correlation between initial template from the standard curve and Ct values, we measured 7.69×102 copies viable cells per 100 mg sick leaves. This study showed that PMA-q PCR method could quickly identify and quantify the number of living bacteria in actual samples, and provided a technical support for the prevention and control of bacterial angular leaf spot on cucumber.

关 键 词：黄瓜细菌性角斑病 PMA-qPCR 丁香假单胞菌 检测 有活力菌体 

分 类 号：S436.421.1[农业科学—农业昆虫与害虫防治]