Wong Mui-Yun, Smart Christine D
Department of Plant Protection, Faculty of Agriculture, and Institute of Tropical Agriculture, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia.
Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Geneva, NY 14456.
Plant Dis. 2012 Sep;96(9):1365-1371. doi: 10.1094/PDIS-07-11-0593-SR.
A DNA macroarray was previously developed to detect major fungal and oomycete pathogens of solanaceous crops. To provide a convenient alternative for researchers with no access to X-ray film-developing facilities, specific CCD cameras or Chemidoc XRS systems, a chromogenic detection method with sensitivity comparable with chemiluminescent detection, has been developed. A fungal (Stemphylium solani) and an oomycete (Phytophthora capsici) pathogen were used to develop the protocol using digoxigenin (DIG)-labeled targets. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA), including ITS1, 5.8S rDNA, and ITS2, was used as the target gene and polymerase chain reaction amplified as in the previous protocol. Various amounts of species-specific oligonucleotides on the array, quantities of DIG-labeled ITS amplicon, and hybridization temperatures were tested. The optimal conditions for hybridization were 55°C for 2 h using at least 10 pmol of each species-specific oligonucleotide and labeled target at 10 ng/ml of hybridization buffer. Incubation of the hybridized array with anti-DIG conjugated alkaline phosphatase substrates, NBT/BCIP, produced visible target signals between 1 and 3 h compared with 1 h in chemiluminescent detection. Samples from pure cultures, soil, and artificially inoculated plants were also used to compare the detection using chemiluminescent and chromogenic methods. Chromogenic detection was shown to yield similar results compared with chemiluminescent detection in regard to signal specificity, duration of hybridization between the array and targets, and cost, though it takes 1 to 2 h longer for the visualization process, thus providing a convenient alternative for researchers who lack darkroom facilities. To our knowledge, this is the first report of DNA macroarray detection of plant pathogens using a chromogenic method.
先前已开发出一种DNA宏阵列，用于检测茄科作物的主要真菌和卵菌病原体。为了为没有X射线胶片显影设备、特定电荷耦合器件（CCD）相机或化学发光成像系统（Chemidoc XRS系统）的研究人员提供一种便捷的替代方法，已开发出一种灵敏度与化学发光检测相当的显色检测方法。使用地高辛（DIG）标记的靶标，以一种真菌（茄链格孢）和一种卵菌（辣椒疫霉）病原体来制定该方案。核糖体DNA（rDNA）的内部转录间隔区（ITS）区域，包括ITS1、5.8S rDNA和ITS2，用作靶基因，并按照先前的方案进行聚合酶链反应扩增。测试了阵列上各种数量的物种特异性寡核苷酸、DIG标记的ITS扩增子数量以及杂交温度。杂交的最佳条件是在55°C下进行2小时，每种物种特异性寡核苷酸至少使用10 pmol，标记靶标在杂交缓冲液中的浓度为10 ng/ml。与化学发光检测中1小时相比，用抗DIG偶联碱性磷酸酶底物NBT/BCIP孵育杂交后的阵列，在1至3小时内产生可见的靶标信号。还使用来自纯培养物、土壤和人工接种植物的样品来比较化学发光和显色方法的检测效果。显色检测在信号特异性、阵列与靶标之间的杂交持续时间和成本方面与化学发光检测显示出相似的结果，尽管可视化过程需要多1至2小时，从而为缺乏暗室设施的研究人员提供了一种便捷的替代方法。据我们所知，这是首次使用显色方法对植物病原体进行DNA宏阵列检测的报告。
