小麦白粉菌环介导等温扩增检测技术体系的建立与应用

Establishment of loop-mediated isothermal amplification assay for detection of the fungal pathogen Blumeria graminis f.sp.tritici

为建立简便、快速和灵敏的小麦白粉菌Blumeria graminis f.sp.tritici(Bgt)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BGT96224V316_LOCUS1725基因(GenBank:VCU40465.1)为靶标序列,设计并筛选特异性引物,建立小麦白粉菌的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,基于筛选出的1套特异性引物建立的LAMP方法能够从8种白粉菌属菌株和1种腐生菌中特异性地检测到小麦白粉菌;对小麦白粉菌DNA样品的检测灵敏度可达到300 fg/μL;对发病叶片的检测显示该LAMP方法能从人工接种的小麦叶片中准确检测出小麦白粉菌,检出时间为接种4 h及以上。综上,本研究建立了一种小麦白粉菌的LAMP检测方法,具有简便快捷、灵敏度高等特点,丰富了小麦白粉菌的检测体系。

The objective of this study was to develop a simple,rapid and sensitive method for detection of the fungal pathogen Blumeria graminis f.sp.tritici(Bgt)based on loop-mediated isothermal amplification(LAMP).The gene BGT96224V316_LOCUS1725(GenBank:VCU40465.1)was used as the detection target.A set of specific LAMP primers were designed and screened;a LAMP method for rapid detection of Bgt was established,and its specificity,sensitivity,and application effect were evaluated.The specificity test showed that the LAMP method was able to specifically detect Bgt from nine species of plant pathogens,including eight powdery mildew genera and one saprophytic fungus.The sensitivity test showed that the detection limit of the LAMP method was 300 fg/μL genomic DNA.Under artificial inoculation conditions,Bgt could be detected accurately from diseased wheat leaves using the LAMP method 4 h post inoculation or later.In conclusion,this study has established a LAMP detection method for wheat powdery mildew,which is characterized by its simplicity,speediness,and high sensitivity.This method enriches the detection system for wheat powdery mildew.