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

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。

Abstract:

[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.