茄科雷尔氏菌IMSA-LAMP检测方法的建立  

Establishment of IMSA-LAMP detection method for Ralstonia solanacearu

作　　者：袁婷 罗龙辉 张雪吟 刘吉平[1] YUAN Ting;LUO Longhui;ZHANG Xueyin;LIU Jiping(College of Animal Science,South China Agricultural University/Regional Sericulture Training Center For Asia-Pacific,Guangzhou 510642,China)

机构地区：[1]华南农业大学动物科学学院/亚太地区蚕桑培训中心,广东广州510642

出　　处：《华南农业大学学报》2022年第4期89-98,共10页Journal of South China Agricultural University

基　　金：财政部和农业农村部:国家现代农业产业技术体系。

摘　　要：【目的】桑树青枯病是由茄科雷尔氏菌Ralstonia solanacearum引起的一种危害严重的细菌性病害,建立一种快速、灵敏的茄科雷尔氏菌检测方法,对桑树青枯病的有效控制有重要意义。【方法】本研究以茄科雷尔氏菌果胶裂解酶基因(Pectate lyase gene)为靶标,基于等温多自配引发扩增技术(Isothermal multiple self-matchinginitiated amplification,IMSA)的引物设计原理,结合环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)的反应体系,建立一种快速有效检测茄科雷尔氏菌的IMSA-LAMP法,并对该方法的最佳反应参数进行了筛选。【结果】基于果胶裂解酶基因建立的IMSA-LAMP检测方法在64.5℃条件下,45 min内可完成对阳性样品的特异检测,对茄科雷尔氏菌的模板DNA检测灵敏度达200 fg/μL(对应菌为1×10^(2) CFU/mL);对生产上收集的疑似桑树青枯病病样的检出率为87.5%。【结论】IMSA-LAMP检测方法具有良好的实用性,可为桑树青枯病的快速检测、诊断与防疫提供新的技术支持。【Objective】Mulberry bacterial wilt is a seriously harmful bacterial disease caused by Ralstonia solanacearum.Therefore,it is of great significance to establish a rapid and sensitive detection method for R.solanacearum to effectively control mulberry bacterial wilt.【Method】Pectate lyase gene of R.solanacearum was used as the target.Based on the primer design principle of isothermal multiple self-matching-initiated amplification(IMSA),combining the loop-mediated isothermal amplification(LAMP)reaction system,a rapid and effective IMSA-LAMP method for detection of R.solanacearum was established.The optimal reaction parameters of this method were screened.【Result】The IMSA-LAMP method based on pectate lyase gene could complete the specific detection of positive samples within 45 min at 64.5℃,and the detection sensitivity of R.solanacearum template DNA was 200 fg/μL(the corresponding bacteria detection sensitivity was 1×10^(2) CFU/mL);The detection rate of suspected mulberry bacterial wilt samples collected in production was 87.5%.【Conclusion】This method has good practicability and can provide new technical support for the rapid detection,diagnosis and epidemic prevention of mulberry bacterial wilt.

关 键 词：桑树青枯病 IMSA-LAMP 茄科雷尔氏菌 果胶裂解酶基因 

分 类 号：S432.42[农业科学—植物病理学]