Duan Ya-Bing, Ge Chang-Yan, Zhang Xiao-Ke, Wang Jian-Xin, Zhou Ming-Guo
College of Plant Protection, Nanjing Agricultural University, Nanjing, Jiangsu Province, China.
PLoS One. 2014 Oct 20;9(10):e111094. doi: 10.1371/journal.pone.0111094. eCollection 2014.
Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB). The LAMP reaction was optimal at 63 °C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10(-3) ng µL(-1) of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10(-2) ng µL(-1)). Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2%) were confirmed as positive by LAMP, 172 (90.1%) positive by the tissue separation, while 147 (77.0%) positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables.
灰葡萄孢是一种毁灭性的植物病原体，可引发灰霉病。在本研究中，我们基于Bcos5序列，利用羟基萘酚蓝染料（HNB）介导的环介导等温扩增（LAMP）技术，开发了一种灰葡萄孢的可视化检测方法。LAMP反应在63℃下进行45分钟时效果最佳。在扩增前加入HNB后，含有灰葡萄孢DNA的样品在反应后呈现出特征性的天蓝色，而不含DNA或含有其他植物病原真菌DNA的样品则没有。当对LAMP产物进行凝胶电泳时，HNB染色法的结果得到了再次验证。该LAMP检测方法对灰葡萄孢的检测限为每个反应10⁻³ ng μL⁻¹的基因组DNA，比传统PCR（10⁻² ng μL⁻¹）灵敏10倍。在接种的番茄和草莓花瓣中可以检测到灰葡萄孢的接种物。在191份患病样品中，LAMP法确认180份（94.2%）为阳性，组织分离法确认172份（90.1%）为阳性，PCR法确认147份（77.0%）为阳性。由于LAMP检测方法在灵敏度、特异性、重复性、可靠性和可视性方面表现良好，它适用于在储存和运输前对感染植物材料（如切花、水果和蔬菜）中的灰葡萄孢进行快速检测。
