基于Ypt1靶标的大豆疫霉环介导等温扩增技术的研究

大豆疫霉侵染大豆引起的根腐病是大豆生产上的毁灭性病害之一。本研究以Ypt1基因作为靶标,利用环介导等温扩增(LAMP)技术,设计了特异性检测体系,整个过程仅需60 min,即可通过肉眼直接目测检测结果。反应后经浊度仪验证浊度变化、琼脂糖凝胶进行电泳验证和在扩增前加入染料HNB(羟基蔡酚蓝)作为反应指示剂验证扩增结果。特异性检测中,111个大豆疫霉菌株均能产生浊度曲线和扩增到梯形状的条带,同时HNB显色观察到天蓝色的阳性反应,而其它疫霉、腐霉和真菌供试菌株中均没有观察到这些现象;在灵敏度检测中,PsYpt1-LAMP技术最低检测限达到100 pg·μL~(-1),比普通PCR技术的最低检测限高出10倍;在田间应用方面,PsYpt1-LAMP检测技术明显提高了检测效率。本研究建立的LAMP检测体系可用于口岸和田间对大豆疫霉的快速检测。

LAMP assay for rapid detection of Phytophthora sojae based on its Ypt1 gene

Phytophthora sojae is a devastating pathogen that causes soybean Phytophthora root rot. This paper reports the development of a loop-mediated isothermal amplication (LAMP) assay for detection of P. sojae based on Ypt1 gene. The Ypt1-LAMP assay efciently amplied the target gene within 60 min at 64°C and was evaluated for specificity and sensitivity. The specificity of the assay was evaluated against isolates of P. sojae, Phytophthora spp., Pythium spp., and some true fungi. Magnesium pyrophosphate resulting from the LAMP of P. sojae could be detected by real-time measurement of turbidity. Additionally, the DNA products of P. sojae were visualized as a ladder-like banding pattern on a 2% agarose gel electrophoresis, and a positive color (sky blue) was observed only in the presence of P. sojae by addition of hydroxynaphthol blue prior to the amplification, whereas none of the other isolates showed such a color change. The detection limit of Ypt1-specic LAMP assay was 100 pg·μL^-1 of the genomic DNA per reaction, which was up to 10-fold more sensitive than that of the conventional PCR approach. P. sojae from diseased soybean tissues and residues was detected by the LAMP assay and the conventional PCR method. Moreover, the traditional isolation from these samples was also performed using a leaf disk baiting method. The positive-sample ratios were 71/130 (54.6%) by the LAMP assay, 61/130 (46.9%) by the conventional PCR approach, and 50/130 (38.4%) by the leaf disk baiting method, suggesting that the PsYpt1-LAMP assay reported here shows great potential for the rapid visual detection of P. sojae in the field and from the soybean production.