禾谷丝核菌环介导等温扩增检测技术体系的建立

Establishment of loop-mediated isothermal amplification assay for the detection of fungal pathogen Rhizoctonia cerealis

为建立简便、快速和灵敏的小麦纹枯病菌——禾谷丝核菌Rhizoctonia cerealis的分子检测技术体系,基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,以核糖体DNA内转录间隔区(internal transcribed spacer,ITS)为靶标序列,设计并筛选特异性引物,建立禾谷丝核菌的LAMP检测方法,并对其特异性、灵敏度和应用效果进行测定。结果表明,基于筛选获得的4条禾谷丝核菌特异性引物建立的LAMP方法能够从15种植物病原菌中特异性地检出禾谷丝核菌,近缘种立枯丝核菌Rhizoctonia solani和引致早期症状易混淆病害的病原菌禾顶囊壳菌Gaeumannomyces graminis、麦根腐平脐蠕孢菌Bipolaris sorokiniana和禾谷镰孢菌Fusarium graminearum均未检出;该LAMP方法在日光下的灵敏度为10^(-1) ng/μL,在蓝光下的灵敏度为10^(-3) ng/μL;对发病组织的检测显示该LAMP方法能从人工接种的小麦发病组织中准确检测出禾谷丝核菌,检出时间为接种12 h及以上。表明建立的禾谷丝核菌LAMP检测体系特异性好、灵敏度高、稳定可靠。

The objective of this study was to develop a simple,rapid and sensitive method for the detection of fungal pathogen Rhizoctonia cerealis based on loop-mediated isothermal amplification(LAMP).The internal transcribed spacer(ITS)sequences of nuclear ribosomal DNA were used as the detection target.After specific LAMP primers were designed and screened,a LAMP method for rapid detection of R.cerealis was established,and its specificity,sensitivity,and application effect were evaluated.The specificity test showed that the LAMP method was able to specifically detect R.cerealis from 15 species of plant pathogens,including a closely related pathogen(Rhizoctonia solani)and three pathogens(Gaeumannomyces graminis,Bipolaris sorokiniana and Fusarium graminearum)causing similar early symptoms.The sensitivity test showed that the detection limit of the LAMP method was 10^(-3) ng/μL genomic DNA under blue light and 10^(-1) ng/μL genomic DNA under sun light.Under artificially inoculated conditions,R.cerealis could be detected accurately from diseased wheat tissues using the LAMP method at 12 h post inoculation or later.The results indicated that the established LAMP method for rapid detection of R.cerealis was specific,sensitive and reliable.