基于环介导等温扩增技术快速诊断由Fusarium andiyazi引起的水稻恶苗病

Rapid diagnosis of rice bakanae caused by Fusarium andiyazi using a loop-mediated isothermal amplification assay

水稻恶苗病是水稻上的重要病害,在我国各水稻主要种植区均有发生,造成水稻产量的严重损失。Fusarium andiyazi是近年来国外报道的水稻恶苗病的病原菌之一,本研究基于环介导等温扩增技术(loop-mediated isothermal amplification,LAM P),以F.andiyazi的TAT(trichothecene 3-O-acetyltransferase)基因为靶标设计并筛选出一套灵敏、特异的LAM P引物,建立了可快速诊断该病菌所引起的水稻恶苗病的LAMP检测技术。在等温条件下(64℃)只需进行核酸扩增反应80 min,反应前向体系中加入了金属离子指示剂HNB(羟基萘酚蓝),反应后即可肉眼观察反应产物颜色变化判断检测结果,阳性反应呈天蓝色,阴性呈紫色。该TAT-Fan-LAMP技术的最低检测灵敏度为100 pg·μL^(-1)。应用该技术成功地对南京江宁和镇江句容田间采集的由F.andiyazi引起的水稻恶苗病进行快速诊断。该LAMP检测技术的建立为F.andiyazi引起的水稻恶苗病的诊断提供了简便快速的新技术。

Bakanae is an important disease of rice across all major rice-growing regions in China leading to seri- ous rice yield losses. Here we describe a loop-mediated isothermal amplification （LAMP） assay on direct detec- tion of F. andiyazi in the diseased rice tissues, for rapid diagnoses of rice bakanae caused by this pathogen. Based on the sequence of trichothecene 3-O-acetyltransferase （TAT） gene in F. andiyazi, a set of species-specific primers was designed and screened to generate a positive color （ sky blue） in the presence of F. andiyazi with the addition of hydroxynaphthol blue （HNB） indicator prior to amplification, whereas no color change （ pur- ple） in the negative reaction tubes of other species isolates under isothermal condition at 64℃ for 80 min. U- sing the TAT-Fan-LAMP assay with the detection limit at 100 pg · μL-1 genomic DNA for F. andiyazi, we suc- cessfully and rapidly diagnosed the suspected diseased rice plant samples collected from Jiangning, Nanjing and Jurong, Zhenjiang in Jiangsu Province. This method can be used as a new way of diagnosing rice bakanae caused by F. andiyazi.