环介导等温扩增技术检测小苍兰花叶病毒
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摘要
小苍兰花叶病毒Freesia mosaic virus(FreMV)是侵染兰花的主要病毒,严重影响其观赏价值。本研究根据FreMV的外壳蛋白基因序列设计了一组特异性引物,经过一系列条件优化,建立了该病毒的RT-LAMP检测方法。结果显示设计引物能特异扩增FreMV,与其他4种病毒(菜豆黄花叶病毒Bean yellow mosaic virus、黄瓜花叶病毒Cucumber mosaic virus、建兰花叶病毒Cymbidium mosaic virus和齿兰环斑病毒Odontoglossum ringspot virus)不发生反应;该方法灵敏度为RT-PCR的10倍。田间检测20份样品中,RT-LAMP和RT-PCR检测结果一致,检出率为60%。在产物中加入荧光染料SYBR GreenⅠ直接用肉眼观察就可判断样品是否感染FreMV,可省去电泳分析的时间。该方法具有特异性强、灵敏度高、操作简单、快速等特点。
Freesia mosaic virus(FreMV) is one of the main viruses infecting orchids, seriously affecting the ornamental value. In order to establish a reverse transcription loop-mediated isothermal amplification(RT-LAMP)assay, the primers were designed based on the conserved region of the coat protein(CP) gene of FreMV available in GenBank, and the reaction conditions were optimized. FreMV was specifically identified by RT-LAMP by using these primers, but Bean yellow mosaic virus, Cucumber mosaic virus, Cymbidium mosaic virus and Odontoglossum ringspot virus were not amplified. The sensitivity of the RT-LAMP assay was 10-fold higher than that of RT-PCR.The positive rate of 20 samples by using the RT-LAMP was 60%, which was consistent to that of RT-PCR, validating the accuracy of the RT-LAMP assay. In addition,PCR products could be visualized by using SYBR Green Ⅰ, and gel electrophoresis was not a necessity. Hence,this RT-LAMP assay is a specific, sensitive and rapid method for detecting FreMV.
Freesia mosaic virus(FreMV) is one of the main viruses infecting orchids, seriously affecting the ornamental value. In order to establish a reverse transcription loop-mediated isothermal amplification(RT-LAMP)assay, the primers were designed based on the conserved region of the coat protein(CP) gene of FreMV available in GenBank, and the reaction conditions were optimized. FreMV was specifically identified by RT-LAMP by using these primers, but Bean yellow mosaic virus, Cucumber mosaic virus, Cymbidium mosaic virus and Odontoglossum ringspot virus were not amplified. The sensitivity of the RT-LAMP assay was 10-fold higher than that of RT-PCR.The positive rate of 20 samples by using the RT-LAMP was 60%, which was consistent to that of RT-PCR, validating the accuracy of the RT-LAMP assay. In addition,PCR products could be visualized by using SYBR Green Ⅰ, and gel electrophoresis was not a necessity. Hence,this RT-LAMP assay is a specific, sensitive and rapid method for detecting FreMV.
引文
[1] HU J S, FERREIRA S, WANG M, et al. Detection of Cymbidium mosaic virus, Odontoglossum ringspot virus, Tomato spotted wilt virus, and potyviruses infecting orchids in Hawaii [J]. Plant Disease, 1993, 77(5): 464-468.
[2] MENZEL W, JELKMANN W, MAISS E. Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control [J]. Journal of Virological Methods, 2002, 99: 81-92.
[3] THOMPSON J R, WETZEL S, KLERKS M M, et al. Multiplex RT-PCR detection 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.
[4] 梁敏国,刘光华.广东兰花病毒病调查和病原检测[J].江西植保,2004,27(3):97-100.
[5] 柳爱春,赵芸,刘超,等.双重一步法RT-PCR检测2种主要兰花病毒[J].安徽农业科学,2009,37(27):12960-13009.
[6] NOTOMI T, OKAYMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 2000, 28(12): e63.
[7] HARPER S J, WARD L I, CLOVER G R G.Development of LAMP and real-time PCR methods for the rapid detection of Xylella fastidiosa for quarantine and field applications[J]. Phytopathology, 2010, 100(12): 1282-1288.
[8] RANJAN R, KANJAYAN M, SUBRAMANIAM S, et al. Development and evaluation of a one-step reverse transcription-loop mediated isothermal amplification assay (RT-LAMP) for rapid detection of foot and mouth disease virus in India [J]. Virus Disease, 2014, 25(3): 358-364.
[9] SINGH P, MIRDHA B R, AHUJA V, et al. Loop-mediated isothermal amplification(LAMP) assay for rapid detection of Entamoeba histolytica in amoebic liver abscess[J]. World Journal of Microbiology Biotechnology, 2013, 29(1): 27-32.
[10] 高宏伟,徐彪,朱来华,等.应用LAMP检测方法检测肉制品中的单增李斯特菌[J].食品安全质量检测学报,2010,27(1):12-17.
[11] ZHANG Yaoqi, SHAN Xiaoxiao, SHI Lei, et al. Development of a fim Y-based loop-mediated isothermal amplification assay for detection of Salmonella in food [J]. Food Research International, 2012, 45(2): 1011-1015.
[12] 王永,兰青阔,赵新,等. 转基因作物外源转基因成分环介导等温扩增技术检测方法的建立及应用[J]. 中国农业科学, 2009, 42(4): 1473-1477.
[13] CHEN Lili, GUO Jinchao, WANG Qidi, et al. Development of the visual loop-mediated isothermal amplification assay for seven genetically modified maize events and their application in practical samples analysis [J]. Journal of Agricultural and Food Chemistry, 2011, 59(11): 5914-5918.
[2] MENZEL W, JELKMANN W, MAISS E. Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control [J]. Journal of Virological Methods, 2002, 99: 81-92.
[3] THOMPSON J R, WETZEL S, KLERKS M M, et al. Multiplex RT-PCR detection 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.
[4] 梁敏国,刘光华.广东兰花病毒病调查和病原检测[J].江西植保,2004,27(3):97-100.
[5] 柳爱春,赵芸,刘超,等.双重一步法RT-PCR检测2种主要兰花病毒[J].安徽农业科学,2009,37(27):12960-13009.
[6] NOTOMI T, OKAYMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 2000, 28(12): e63.
[7] HARPER S J, WARD L I, CLOVER G R G.Development of LAMP and real-time PCR methods for the rapid detection of Xylella fastidiosa for quarantine and field applications[J]. Phytopathology, 2010, 100(12): 1282-1288.
[8] RANJAN R, KANJAYAN M, SUBRAMANIAM S, et al. Development and evaluation of a one-step reverse transcription-loop mediated isothermal amplification assay (RT-LAMP) for rapid detection of foot and mouth disease virus in India [J]. Virus Disease, 2014, 25(3): 358-364.
[9] SINGH P, MIRDHA B R, AHUJA V, et al. Loop-mediated isothermal amplification(LAMP) assay for rapid detection of Entamoeba histolytica in amoebic liver abscess[J]. World Journal of Microbiology Biotechnology, 2013, 29(1): 27-32.
[10] 高宏伟,徐彪,朱来华,等.应用LAMP检测方法检测肉制品中的单增李斯特菌[J].食品安全质量检测学报,2010,27(1):12-17.
[11] ZHANG Yaoqi, SHAN Xiaoxiao, SHI Lei, et al. Development of a fim Y-based loop-mediated isothermal amplification assay for detection of Salmonella in food [J]. Food Research International, 2012, 45(2): 1011-1015.
[12] 王永,兰青阔,赵新,等. 转基因作物外源转基因成分环介导等温扩增技术检测方法的建立及应用[J]. 中国农业科学, 2009, 42(4): 1473-1477.
[13] CHEN Lili, GUO Jinchao, WANG Qidi, et al. Development of the visual loop-mediated isothermal amplification assay for seven genetically modified maize events and their application in practical samples analysis [J]. Journal of Agricultural and Food Chemistry, 2011, 59(11): 5914-5918.