Lievens Bart, Brouwer Margreet, Vanachter Alfons C R C, Lévesque C André, Cammue Bruno P A, Thomma Bart P H J
Centre of Microbial and Plant Genetics (CMPG), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Heverlee-Leuven, Belgium.
Environ Microbiol. 2005 Nov;7(11):1698-710. doi: 10.1111/j.1462-2920.2005.00816.x.
Detection, identification and quantification of plant pathogens are the cornerstones of preventive plant disease management. To detect multiple pathogens in a single assay, DNA array technology currently is the most suitable technique. However, for sensitive detection, polymerase chain reaction (PCR) amplification before array hybridization is required. To evaluate whether DNA array technology can be used to simultaneously detect and quantify multiple pathogens, a DNA macroarray was designed and optimized for accurate quantification over at least three orders of magnitude of the economically important vascular wilt pathogens Verticillium albo-atrum and Verticillium dahliae. A strong correlation was observed between hybridization signals and pathogen concentrations for standard DNA added to DNA from different origins and for infested samples. While accounting for specific criteria like amount of immobilized detector oligonucleotide and controls for PCR kinetics, accurate quantification of pathogens was achieved in concentration ranges typically encountered in horticultural practice. Subsequently, quantitative assessment of other tomato pathogens (Fusarium oxysporum, Fusarium solani, Pythium ultimum and Rhizoctonia solani) in environmental samples was performed using DNA array technology and correlated to measurements obtained using real-time PCR. As both methods of quantification showed a very high degree of correlation, the reliability and robustness of the DNA array technology is shown.
植物病原体的检测、鉴定和定量是植物病害预防管理的基石。要在一次检测中检测多种病原体，DNA阵列技术是目前最合适的技术。然而，为了进行灵敏检测，在阵列杂交之前需要进行聚合酶链反应（PCR）扩增。为了评估DNA阵列技术是否可用于同时检测和定量多种病原体，设计并优化了一种DNA宏阵列，用于对经济上重要的维管束萎蔫病原体黑白轮枝菌和大丽轮枝菌在至少三个数量级上进行准确定量。对于添加到来自不同来源的DNA中的标准DNA以及受侵染的样品，杂交信号与病原体浓度之间观察到很强的相关性。在考虑诸如固定化检测寡核苷酸的量和PCR动力学的对照等特定标准的情况下，在园艺实践中通常遇到的浓度范围内实现了病原体的准确定量。随后，使用DNA阵列技术对环境样品中的其他番茄病原体（尖孢镰刀菌、茄腐镰刀菌、终极腐霉和立枯丝核菌）进行了定量评估，并与使用实时PCR获得的测量结果相关联。由于两种定量方法都显示出非常高的相关性，因此证明了DNA阵列技术的可靠性和稳健性。
