中国香菇种质资源中主要真菌病毒的多重RT-PCR检测技术  

Development of multiplex RT-PCR for the detection of two main mycoviruses infecting Chinese Lentinula edodes germplasm resource

作　　者：孙艺嘉 郭孟配 王锦杰 边银丙[1,2] 徐章逸 SUN Yi-Jia;GUO Meng-Pei;WANG Jin-Jie;BIAN Yin-Bing;XU Zhang-Yi(Institute of Applied Mycology, Huazhong Agricultural University, Wuhan, Hubei 430070, China;Hubei Engineering Research Center for Edible Mushroom, Wuhan, Hubei 430070, China)

机构地区：[1]华中农业大学应用真菌研究所,湖北武汉430070 [2]湖北省食用菌工程技术研究中心,湖北武汉430070

出　　处：《微生物学通报》2019年第6期1381-1389,共9页Microbiology China

基　　金：国家现代农业产业技术体系“食用菌病害无害化轻简化防控”岗位科学家专项(CARS-20);农业部华中作物有害生物综合治理重点实验室/农作物重大病虫草害防控湖北省重点实验室开放基金(2015ZTSJJ11)~~

摘　　要：【背景】病毒是食用菌生产上较重要的一类潜在隐患。我们在前期的研究中发现,中国香菇(Lentinula edodes)种质资源中最常见的病毒有2种,分别为L. edodes partitivirus 1 (LePV1)和L. edodes mycovirus HKB (LeV-HKB)病毒,这2种病毒能单一或复合感染香菇。【目的】建立香菇种质主要病毒的快速检测技术,提高香菇病毒检测效率,降低检测成本。【方法】依据香菇β-actin基因及病毒LePV1和LeV-HKB编码依赖RNA的RNA复制酶(RdRp)基因序列分别设计引物,以复合感染了这2种香菇病毒的菌丝体为材料,对影响RT-PCR反应的主要因素模板浓度、引物浓度、dNTPs浓度、退火温度和循环次数等进行优化筛选,建立同时检测LeV-HKB病毒(LeV-HKB相关病毒)和LePV1病毒的多重RT-PCR检测技术。【结果】模板浓度、退火温度和循环次数对多重RT-PCR检测结果均有较大影响,而引物浓度和dNTPs浓度对检测结果的影响较小。所建立的多重RT-PCR技术在香菇核心种质病毒检测中表现为扩增目标条带清晰分明,检测结果与单重RT-PCR检测结果一致。对两种病毒的多重RT-PCR产物进行了序列测序,结果表明扩增片段序列与目标基因序列相似性在99%以上。【结论】所建立的多重RT-PCR检测体系具有较好的特异性与适用性,为室内和田间香菇病毒早期检测、病害流行的监测提供了技术储备。[Background] Lentinula edodes virus disease is important for L. edodes production. Previous to this study, L. edodes mycovirus HKB(LeV-HKB) and L. edodes partitivirus 1(LePV1) were proved to be the two major mycoviruses identified in L. edodes germplasm, and co-infection of these two viruses was also prevalent.[Objective] The purpose of this study was to develop a quick and efficient technique for detection of these two viruses in the early stage of L. edodes production.[Methods] Three pairs of specific primers for LeV-HKB, LePV1 and actin genes(β-actin) of L. edodes were designed based on the reported sequences, respectively. The crucial factors of multiplex RT-PCR including template concentration,primers concentration, dNTPs concentration, annealing temperature, and number of cycles were then optimized.[Results] A multiplex RT-PCR detection method for LeV-HKB(or LeV-HKB related virus) and LePV1 was developed.[Conclusion] The multiplex RT-PCR assay developed in this study had high specificity and repeatability using the virus detection of L. edodes core collections.

关 键 词：香菇 食用菌 真菌病毒 检测 

分 类 号：S436.46[农业科学—农业昆虫与害虫防治]