环介导等温扩增技术快速检测麦根腐平脐蠕孢  

Rapid detection of Bipolaris sorokiniana based on loop-mediated isothermal amplification

作　　者：崔林开[1] 和志华 康业斌[1] 胡艳红[1] CUI Linkai;HE Zhihua;KANG Yebin;HU Yanhong(College of Horticulture and Plant Protection,Henan University of Science and Technology,Luoyang 471023,China)

机构地区：[1]河南科技大学园艺与植物保护学院,洛阳471023

出　　处：《植物病理学报》2022年第2期269-275,共7页Acta Phytopathologica Sinica

基　　金：国家自然科学基金项目(U1804104);河南省科技攻关计划项目(212102110141)。

摘　　要：为建立麦根腐平脐蠕孢简单快速的分子检测方法,基于环介导等温扩增(Loop-mediated isothermal amplification,LAMP)技术,以核糖体DNA的ITS为靶标序列,设计和筛选出一组麦根腐平脐蠕孢的特异性引物,成功开发可快速准确检测麦根腐平脐蠕孢的LAMP方法。甜菜碱作用测试显示LAMP体系中是否添加甜菜碱对扩增结果无明显影响;特异性测试显示该LAMP方法能够从14种植物病原菌中特异地检测出麦根腐平脐蠕孢;灵敏度测试显示该LAMP方法的检测极限为10^(-3)ng·μL^(-1),是常规PCR检测方法灵敏度的100倍;通用性测试显示该LAMP方法能够准确检测洛阳、安阳、开封和邯郸等不同地理来源的麦根腐平脐蠕孢菌株;发病组织检测显示该LAMP方法能够准确地从人工接种的小麦发病组织中检测出麦根腐平脐蠕孢,检出时间为侵染12 h及以上。这些研究结果表明所建立的麦根腐平脐蠕孢LAMP检测方法特异性好、灵敏度高、适用性强,可用于小麦根腐病的早期快速诊断。The objective of this study was to develop a simple and rapid method for detection of Bipolaris sorokiniana by loop-mediated isothermal amplification(LAMP).LAMP primers were designed based on the internal transcribed spacer(ITS)sequence of nuclear ribosomal DNA.After a set of specific LAMP primers was screened from the designed primers,a method for rapid and accurate detection of B.sorokiniana was established successfully.The effect test for betafin showed that amplification systems with and without betafin has no obvious effects on amplification results.The specificity test showed that the LAMP method was able to specifically detect B.sorokiniana from 14 species of plant pathogens.The sensitivity test showed that the detection limit of the LAMP method was 10^(-3)ng·μL^(-1),which was 100 times higher than that of the conventional PCR detection.The utility test showed that the LAMP method was able to accurately detect B.sorokiniana isolates from different regions,including those collected from Luoyang,Anyang,Kaifeng and Handan.Under artificially inoculated conditions,B.sorokiniana could be detected accurately by the LAMP method from diseased wheat tissues at 12 h post inoculation or longer.The results indicated that the developed LAMP method was sensitive,specific and reliable,and it was suitable for early diagnosis of wheat root rot caused by B.sorokiniana.

关 键 词：小麦根腐病 麦根腐平脐蠕孢 环介导等温扩增 快速检测 

分 类 号：S432.44[农业科学—植物病理学]