Plant Health and Environment Laboratory, Investigation and Diagnostic Centre, MAF Biosecurity New Zealand, P.O. Box 2095, Auckland 1140, New Zealand.
Phytopathology. 2010 Dec;100(12):1282-8. doi: 10.1094/PHYTO-06-10-0168.
Xylella fastidiosa is a regulated plant pathogen in many parts of the world. To increase diagnostic capability of X. fastidiosa in the field, a loop-mediated isothermal amplification (LAMP) and real-time polymerase chain reaction (PCR) assay were developed to the rimM gene of X. fastidiosa and evaluated for specificity and sensitivity. Both assays were more robust than existing published assays for detection of X. fastidiosa when screened against 20 isolates representing the four major subgroups of the bacterium from a range of host species. No cross-reaction was observed with DNA from healthy hosts or other bacterial species. The LAMP and real-time assays could detect 250 and 10 copies of the rimM gene, respectively, and real-time sensitivity was comparable with an existing published real-time PCR assay. Hydroxynapthol blue was evaluated as an endpoint detection method for LAMP. When at least 500 copies of target template were present, there was a noticeable color change indicating the presence of the bacterium. Techniques suitable for DNA extraction from plant tissue in situ were compared with a standard silica-column-based laboratory extraction method. A portable PickPen and magnetic bead system could be used to successfully extract DNA from infected tissue and could be used in conjunction with LAMP in the field.
韧皮部难养菌是世界上许多地区的管制性植物病原体。为了提高韧皮部难养菌在田间的诊断能力，开发了针对韧皮部难养菌 rimM 基因的环介导等温扩增（LAMP）和实时聚合酶链反应（PCR）检测方法，并对其特异性和灵敏度进行了评估。在对来自不同宿主物种的四种主要细菌亚群的 20 个分离株进行筛选时，这两种检测方法都比现有的发表的检测方法更稳健。与来自健康宿主或其他细菌的 DNA 没有交叉反应。LAMP 和实时检测分别可以检测到 250 和 10 个 rimM 基因拷贝，实时灵敏度与现有的发表的实时 PCR 检测相当。羟基萘蓝被评估为 LAMP 的终点检测方法。当存在至少 500 个拷贝的目标模板时，会发生明显的颜色变化，表明存在细菌。比较了适合从原位植物组织中提取 DNA 的技术与基于标准硅胶柱的实验室提取方法。便携式 PickPen 和磁珠系统可成功地从感染组织中提取 DNA，并可与 LAMP 一起在现场使用。
