中国香菇种质资源中主要真菌病毒的多重RT-PCR检测技术
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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.
[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.
引文
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[14]Pei GQ,Dong YF,Zhang ZP,et al.Detection of four grapevine leafroll-associated viruses by multiplex RT-PCR[J].Acta Phytopathologica Sinica,2010,40(1):21-26(in Chinese)裴光前,董雅凤,张尊平,等.4种葡萄卷叶伴随病毒多重RT-PCR检测[J].植物病理学报,2010,40(1):21-26
[15]Jiang SH,Cheng XM,Song CX,et al.Establishment of multiplex RT-PCR system for detection of three viruses in sweet potato[J].Plant Protection,2017,43(1):126-130(in Chinese)蒋素华,程喜梅,宋彩霞,等.三种甘薯病毒多重RT-PCR检测技术的建立[J].植物保护,2017,43(1):126-130
[16]Wang JJ,Guo MP,Sun YJ,et al.Detection of the occurrence of two major viruses Lentinula edodes mycovirus HKB and L.edodes partitivirus 1 in Chinese L.edodes germplasm resources[J].Mycosystema,2018,37(4):522-537(in Chinese)王锦杰,郭孟配,孙艺嘉,等.中国香菇种质资源中两种主要病毒的携带情况检测[J].菌物学报,2018,37(4):522-537
[17]Xiao Y,Cheng XJ,Liu J,et al.Population genomic analysis uncovers environmental stress-driven selection and adaptation of Lentinula edodes population in China[J].Scientific Reports,2016,6:36789
[18]Shui PR,Zheng XB,Lin JF,et al.A rapid and efficient method for isolating high quality total RNA from edible fungi[J].Acta Edulis Fungi,2008,15(1):32-36税丕容,郑晓冰,林俊芳,等.简便高质量的食用菌总RNA提取方法[J].食用菌学报,2008,15(1):32-36
[19]Sahin E,Akata I.Viruses infecting macrofungi[J].Virusdisease,2018,29(1):1-18
[20]Wu XP,Wang L,Xie BG,et al.Studies on virus-free strain of Lentinula edodes[J].Chinese Agricultural Science Bulletin,2009,25(24):44-49(in Chinese)吴小平,王丽,谢宝贵,等.香菇菌株的脱毒研究[J].中国农学通报,2009,25(24):44-49
[2]Ushiyama R,Nakai Y,Ikegami M.Evidence for double-stranded RNA from polyhedral virus-like particles in Lentinus edodes(Berk.)Sing[J].Virology,1977,77(2):880-883
[3]Yu SQ,Wang MQ,Zhang RP,et al.Studies on Lentinus edodes virus diseasesⅠ.The occurrence of Lentinus edodes viruses in China[J].Acta Mycologica Sinica,1985,4(2):125-129(in Chinese)于善谦,王鸣岐,张若平,等.香菇病毒的研究Ⅰ.发生在我国的香菇病毒[J].真菌学报,1985,4(2):125-129
[4]Shen XR,Shen JY,Chen ZY,et al.A single-stranded virus isolated from Shiitake mushroom Lentinus edodes(Berk.)Sing[J].Virologica Sinica,1992,7(1):99-105(in Chinese)沈学仁,沈菊英,陈作义,等.一种含有单链RNA的香菇球状病毒[J].中国病毒学,1992,7(1):99-105
[5]Liang ZP,Zhang XX,Gao P,et al.Two kind viruses isolated from fruitbody of Lentinus edodes[J].Edible Fungi of China,2005,24(6):32-33(in Chinese)梁振普,张小霞,高鹏,等.从香菇子实体中分离了两种病毒[J].中国食用菌,2005,24(6):32-33
[6]Yao L,Chen CL,Zhang ZX,et al.The partial genome cDNAsequence of a novel dsRNA virus from Lentinus edodes and the virus detected by RT-PCR[J].Microbiology China,2010,37(1):61-70(in Chinese)姚立,陈春乐,张忠信,等.一种新香菇病毒基因组部分c DNA序列及病毒RT-PCR检测[J].微生物学通报,2010,37(1):61-70
[7]Magae Y.Molecular characterization of a novel mycovirus in the cultivated mushroom,Lentinula edodes[J].Virology Journal,2012,9(1):60
[8]Won HK,Park SJ,Kim DK,et al.Isolation and characterization of a mycovirus in Lentinula edodes[J].Journal of Microbiology,2013,51(1):118-122
[9]Kim JM,Jung JE,Park JA,et al.Biological function of a novel chrysovirus,CnV1-BS122,in the Korean Cryphonectria nitschkei BS122 strain[J].Journal of Bioscience and Bioengineering,2013,115(1):1-3
[10]Ohta C,Taguchi T,Takahashi S,et al.Detection of double stranded RNA elements in cultivated Lentinula edodes[J].Mushroom Science and Biotechnology,2008,16:155-158
[11]Guo MP,Bian YB,Wang JJ,et al.Biological and molecular characteristics of a novel partitivirus infecting the edible fungus Lentinula edodes[J].Plant Disease,2017,101(5):726-733
[12]Chen CL,Zhang ZX,Ying GH,et al.Detection of a rod-shaped virus in Lentinula edodes mycelium using RT-PCR[J].Acta Edulis Fungi,2011,18(3):79-82(in Chinese)陈春乐,张忠信,应国华,等.香菇菌丝体杆形病毒的RT-PCR检测及鉴定[J].食用菌学报,2011,18(3):79-82
[13]Thompson JR,Wetzel S,Klerks MM,et al.Multiplex RT-PCRdetection of four aphid-borne strawberry viruses in Fragaria spp.in combination with a plant mRNA specific internal control[J].Journal of Virological Methods,2003,111(2):85-93
[14]Pei GQ,Dong YF,Zhang ZP,et al.Detection of four grapevine leafroll-associated viruses by multiplex RT-PCR[J].Acta Phytopathologica Sinica,2010,40(1):21-26(in Chinese)裴光前,董雅凤,张尊平,等.4种葡萄卷叶伴随病毒多重RT-PCR检测[J].植物病理学报,2010,40(1):21-26
[15]Jiang SH,Cheng XM,Song CX,et al.Establishment of multiplex RT-PCR system for detection of three viruses in sweet potato[J].Plant Protection,2017,43(1):126-130(in Chinese)蒋素华,程喜梅,宋彩霞,等.三种甘薯病毒多重RT-PCR检测技术的建立[J].植物保护,2017,43(1):126-130
[16]Wang JJ,Guo MP,Sun YJ,et al.Detection of the occurrence of two major viruses Lentinula edodes mycovirus HKB and L.edodes partitivirus 1 in Chinese L.edodes germplasm resources[J].Mycosystema,2018,37(4):522-537(in Chinese)王锦杰,郭孟配,孙艺嘉,等.中国香菇种质资源中两种主要病毒的携带情况检测[J].菌物学报,2018,37(4):522-537
[17]Xiao Y,Cheng XJ,Liu J,et al.Population genomic analysis uncovers environmental stress-driven selection and adaptation of Lentinula edodes population in China[J].Scientific Reports,2016,6:36789
[18]Shui PR,Zheng XB,Lin JF,et al.A rapid and efficient method for isolating high quality total RNA from edible fungi[J].Acta Edulis Fungi,2008,15(1):32-36税丕容,郑晓冰,林俊芳,等.简便高质量的食用菌总RNA提取方法[J].食用菌学报,2008,15(1):32-36
[19]Sahin E,Akata I.Viruses infecting macrofungi[J].Virusdisease,2018,29(1):1-18
[20]Wu XP,Wang L,Xie BG,et al.Studies on virus-free strain of Lentinula edodes[J].Chinese Agricultural Science Bulletin,2009,25(24):44-49(in Chinese)吴小平,王丽,谢宝贵,等.香菇菌株的脱毒研究[J].中国农学通报,2009,25(24):44-49
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