Feng Wenzhuo, Ishiguro Yasushi, Hotta Keisuke, Watanabe Hideki, Suga Haruhisa, Kageyama Koji
Graduate School of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
River Basin Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
FEMS Microbiol Lett. 2015 Nov;362(21). doi: 10.1093/femsle/fnv174. Epub 2015 Sep 22.
Pythium irregulare is an important soil-borne pathogen that causes seed, stem and root rot, and seedling damping-off in various crops. Here, we have developed a rapid and reliable approach for detecting the pathogen using loop-mediated isothermal amplification (LAMP) in combination with primers designed from the sequences of the P. irregulare ribosomal DNA internal transcribed spacer region. The specificity of the primers for P. irregulare was tested using 50 isolates of 40 Pythium species, 11 Phytophthora isolates and 8 isolates of 7 other soil-borne pathogens. The assay showed that the limit of sensitivity of the LAMP method was 100 fg of pure DNA, a similar level to that of a polymerase chain reaction. LAMP detected P. irregulare from the supernatant after mixing culture medium (template DNA source) with distilled water. Similarly, positive results were obtained using a 'Plant-LAMP' method applied to a suspension rotted roots in water. A 'Bait-LAMP' method using the supernatant of autoclaved perilla seeds incubated in a soil/water mixture for 1 week at 25°C successfully detected P. irregulare from the soil. The LAMP assay described in this study is therefore a simple and effective way for practical detection of P. irregulare.
不规则腐霉是一种重要的土传病原菌，可导致多种作物发生种子、茎和根腐烂以及幼苗猝倒病。在此，我们开发了一种快速可靠的方法，利用环介导等温扩增技术（LAMP）并结合根据不规则腐霉核糖体DNA内转录间隔区序列设计的引物来检测该病原菌。使用40种腐霉属的50个分离株、11个疫霉属分离株以及7种其他土传病原菌的8个分离株，对不规则腐霉引物的特异性进行了测试。该检测方法表明，LAMP方法的灵敏度极限为100 fg纯DNA，与聚合酶链反应的灵敏度水平相似。LAMP可从将培养基（模板DNA来源）与蒸馏水混合后的上清液中检测到不规则腐霉。同样，将“植物LAMP”方法应用于水中腐烂根系的悬浮液时也获得了阳性结果。使用在25°C下于土壤/水混合物中孵育1周的高压灭菌紫苏种子的上清液的“诱饵LAMP”方法成功从土壤中检测到了不规则腐霉。因此，本研究中描述的LAMP检测方法是实际检测不规则腐霉的一种简单有效的方法。
