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《工程(英文)》 >> 2021年 第7卷 第9期 doi: 10.1016/j.eng.2020.07.016

利用基因组特异性引物和Cas12a介导技术快速检测稻瘟菌小麦致病型

a State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
b Department of Plant Pathology, Ohio State University, Columbus, OH 43210, USA
c Institute of Biotechnology and Genetic Engineering (IBGE), Bangabandhu Sheikh Mujibur Rahman Agricultural University, Dhaka 1706, Bangladesh
d Centro de Ciências Agrárias, Universidade Federal de São Carlos, Araras, SP CEP 13600-000, Brazil
e United States Department of Agriculture-Agricultural Research Service (USDA-ARS), Foreign Disease-Weed Science Research Unit (FDWSRU), Ft. Detrick, MD 21702, USA

# These authors contributed equally to this work.

收稿日期: 2020-03-30 修回日期: 2020-06-03 录用日期: 2020-07-20 发布日期: 2020-09-10

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摘要

麦瘟病由稻瘟菌小麦致病型(MoT)引起,是一种存在于南美洲和孟加拉国的毁灭性病害。由于MoT通常不会在抽穗期之前使小麦显现染病症状,但在抽穗期其侵染会一直加剧,因此仅根据肉眼观察来使用杀菌剂是无效的。为了开发一种在苗期和营养期准确、灵敏检测MoT以控制病害蔓延的方法,我们对来自巴西的两个MoT分离株的基因组进行了测序,并确定了两个DNA片段:MoT-6098MoT-6099,它们存在于麦瘟病菌(MoT)基因组中,但不存在于感染稻瘟病菌(MoO)的水稻基因组中。利用聚合酶链反应(PCR),我们在来自南美洲和孟加拉国的53 株MoT和MoO分离株中证实了这两对标记引物的特异性。为了测试这两对标记引物的有效性,我们首先建立了一种环介导等温扩增(LAMP)方法。在等温条件下,这种方法在不使用常规PCR仪器的情况下也能检测MoT。随后,我们使用Cas12a 蛋白及导向RNA(gRNA)靶向MoT-6098MoT-6099 序列,两个靶标被识别后,均能激活Cas12a 非特异切割单链脱氧核糖核酸酶(ssDNAase)的活性,我们将依赖靶向Cas12a 激活ssDNase 活性,同时将重组酶聚合酶扩增(RPA)和核酸侧流免疫分析法(NALFIA)相结合,开发出一种准确、灵敏且经济高效地检测受感染小麦植株中MoT特异性DNA序列的方法。这种新技术可应用于田间麦瘟病和其他重要植物病害的快速检测。

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