桑脉带相关病毒RT-LAMP检测方法的建立
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摘要
桑脉带相关病毒Mulberry vein banding-associated virus (MVBaV)是广西桑树病毒病的主要病原病毒。本研究根据MVBaV基因组的核外壳蛋白(nucleocapsid protein, N)基因序列设计了5组引物,筛选获得了1组有效引物,建立了该病毒的反转录环介导等温扩增技术(reverse transcription loop-mediated isothermal amplification, RT-LAMP)检测方法。该方法能在恒温条件(63℃)下1 h内检测MVBaV,其灵敏度是RT-PCR检测方法的10倍。对6个田间样品进行RT-LAMP检测,其结果与RT-PCR的结果一致。该方法可直接在反应管中加入SYBR GreenⅠ,通过颜色变化即可判定结果,无需经过琼脂糖电泳或专门仪器,具有灵敏、快速和特异性好的优点,可应用于MVBaV的快速诊断及其田间监测。
Mulberry vein banding-associated virus(MVBaV) is one of the main pathogenic viruses infecting mulberry in Guangxi. Five sets of specific primers were designed based on MVBaV nucleocapsid protein(N) gene for reverse transcription loop-mediated isothermal amplification(RT-LAMP) assay, and one feasible set of primers was selected. The RT-LAMP method was developed for the detection of MVBaV from mulberry. In the RT-LAMP method, MVBaV genomic RNA could be amplified under isothermal conditions(63℃) within 1 h, and was 10 times more sensitive than RT-PCR. Consistent results of six mulberry samples from the field were obtained by the RT-LAMP and RT-PCR assay, suggesting the reliability of RT-LAMP in MVBaV detection.The results were validated by the measurement or observation of the color of the reaction mixture after adding SYBR GreenⅠwithout gel electrophoresis or special equipments. This RT-LAMP method enabled sensitive, rapid and specific detection of MVBaV and could be applied for the rapid diagnosis and surveillance of MVBaV in the field.
Mulberry vein banding-associated virus(MVBaV) is one of the main pathogenic viruses infecting mulberry in Guangxi. Five sets of specific primers were designed based on MVBaV nucleocapsid protein(N) gene for reverse transcription loop-mediated isothermal amplification(RT-LAMP) assay, and one feasible set of primers was selected. The RT-LAMP method was developed for the detection of MVBaV from mulberry. In the RT-LAMP method, MVBaV genomic RNA could be amplified under isothermal conditions(63℃) within 1 h, and was 10 times more sensitive than RT-PCR. Consistent results of six mulberry samples from the field were obtained by the RT-LAMP and RT-PCR assay, suggesting the reliability of RT-LAMP in MVBaV detection.The results were validated by the measurement or observation of the color of the reaction mixture after adding SYBR GreenⅠwithout gel electrophoresis or special equipments. This RT-LAMP method enabled sensitive, rapid and specific detection of MVBaV and could be applied for the rapid diagnosis and surveillance of MVBaV in the field.
引文
[1] 张璐,朱丽玲,潘瑞兰,等.利用酵母双杂交系统研究桑脉带相关病毒核外壳蛋白自身相互作用[J].基因组学与应用生物学,2015,34(11):2428-2435.
[2] 邱长玉,朱方容,林强.桑树花叶病的研究进展[J].广西蚕业,2011(2):32-37.
[3] MENG Jiaorong, LIU Pingping, ZHU Liling, et al. Complete genome sequence of mulberry vein banding associated virus, a new tospovirus infecting mulberry [J/OL]. PLoS ONE, 2015, 10(8): e0136196.
[4] MENG Jiaorong, LIU Pingping, ZOU Chengwu, et al. First report of a tospovirus in mulberry [J]. Plant Disease, 2013, 97(7): 1001.
[5] 陶源, 吴兴泉. 植物病毒检测方法的研究进展[J]. 分子植物育种, 2017, 15(7): 2901-2906.
[6] FANG Y, RAMASAMY R P. Current and prospective methods for plant disease detection[J]. Biosensors, 2015, 5(3): 537-561.
[7] BOONHAM N, KREUZE J, WINTER S, et al. Methods in virus diagnostics: from ELISA to next generation sequencing[J]. Virus Research, 2014, 186: 20-31.
[8] WARD L I, HARPER S J. Loop-mediated isothermal amplification for the detection of plant pathogens [M]//SUCHER N J, HENNELL J R, CARLES M C. Plant DNA Fingerprinting and Barcoding: Methods and Protocols.New York: Humana Press, 2012: 161-170.
[9] NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J/OL]. Nucleic Acids Research, 2000, 28(12): e63.
[10] WU Xinghai, CHEN Chanfa, XIAO Xizhi, et al. Development of reverse transcription thermostable helicase-dependent DNA amplification for the detection of tomato spotted wilt virus [J]. Journal of AOAC International, 2016, 99(6): 1596-1599.
[11] 张永江, 辛言言, 李桂芬, 等. 葡萄A病毒RT-LAMP检测方法的建立[J].中国农业科学, 2016, 49(1): 103-109.
[12] 汤亚飞, 何自福, 佘小漫, 等. 辣椒黄脉病毒RT-LAMP快速检测方法的建立[J].植物保护, 2016, 42(6): 100-104.
[13] 陈柳, 尚巧霞, 陈笑瑜, 等. 草莓轻型黄边病毒RT-LAMP检测方法的建立[J].中国农业科学, 2015, 48(3): 613-620.
[14] 陈先锋,张吉红,崔俊霞,等.南芥菜花叶病毒的RT-LAMP检测试剂盒的研制[J].植物保护学报,2013,40(2):189-190.
[15] 郭木金,廖富荣, 陈青, 等. 豇豆重花叶病毒RT-LAMP检测方法的建立[J].植物保护, 2012, 38(1): 123-127.
[16] 蒯元璋. 桑树病毒与病毒病的研究进展(Ⅰ)[J].蚕业科学, 2010(5): 818-825.
[17] 陈明胜, 吴福安. 桑花叶病研究的现状与对策[J].中国蚕业, 2009(2): 20-23.
[18] MA Yuxin, NAVARRO B, ZHANG Zhixiang, et al. Identification and molecular characterization of a novel monopartite geminivirus associated with mulberry mosaic dwarf disease [J].The Journal of General Virology,2015,96(8):2421-2434.
[19] CHIUMENTI M, MORELLI M, DE STRADIS A, et al. Unusual genomic features of a badnavirus infecting mulberry [J]. The Journal of General Virology, 2016, 97(11): 3073-3087.
[20] LU Quanyou, WU Zujian, XIA Zhisong, et al. A new nepovirus identified in mulberry (Morus alba L.) in China [J]. Archives of Virology, 2015, 160(3): 851-855.
[2] 邱长玉,朱方容,林强.桑树花叶病的研究进展[J].广西蚕业,2011(2):32-37.
[3] MENG Jiaorong, LIU Pingping, ZHU Liling, et al. Complete genome sequence of mulberry vein banding associated virus, a new tospovirus infecting mulberry [J/OL]. PLoS ONE, 2015, 10(8): e0136196.
[4] MENG Jiaorong, LIU Pingping, ZOU Chengwu, et al. First report of a tospovirus in mulberry [J]. Plant Disease, 2013, 97(7): 1001.
[5] 陶源, 吴兴泉. 植物病毒检测方法的研究进展[J]. 分子植物育种, 2017, 15(7): 2901-2906.
[6] FANG Y, RAMASAMY R P. Current and prospective methods for plant disease detection[J]. Biosensors, 2015, 5(3): 537-561.
[7] BOONHAM N, KREUZE J, WINTER S, et al. Methods in virus diagnostics: from ELISA to next generation sequencing[J]. Virus Research, 2014, 186: 20-31.
[8] WARD L I, HARPER S J. Loop-mediated isothermal amplification for the detection of plant pathogens [M]//SUCHER N J, HENNELL J R, CARLES M C. Plant DNA Fingerprinting and Barcoding: Methods and Protocols.New York: Humana Press, 2012: 161-170.
[9] NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J/OL]. Nucleic Acids Research, 2000, 28(12): e63.
[10] WU Xinghai, CHEN Chanfa, XIAO Xizhi, et al. Development of reverse transcription thermostable helicase-dependent DNA amplification for the detection of tomato spotted wilt virus [J]. Journal of AOAC International, 2016, 99(6): 1596-1599.
[11] 张永江, 辛言言, 李桂芬, 等. 葡萄A病毒RT-LAMP检测方法的建立[J].中国农业科学, 2016, 49(1): 103-109.
[12] 汤亚飞, 何自福, 佘小漫, 等. 辣椒黄脉病毒RT-LAMP快速检测方法的建立[J].植物保护, 2016, 42(6): 100-104.
[13] 陈柳, 尚巧霞, 陈笑瑜, 等. 草莓轻型黄边病毒RT-LAMP检测方法的建立[J].中国农业科学, 2015, 48(3): 613-620.
[14] 陈先锋,张吉红,崔俊霞,等.南芥菜花叶病毒的RT-LAMP检测试剂盒的研制[J].植物保护学报,2013,40(2):189-190.
[15] 郭木金,廖富荣, 陈青, 等. 豇豆重花叶病毒RT-LAMP检测方法的建立[J].植物保护, 2012, 38(1): 123-127.
[16] 蒯元璋. 桑树病毒与病毒病的研究进展(Ⅰ)[J].蚕业科学, 2010(5): 818-825.
[17] 陈明胜, 吴福安. 桑花叶病研究的现状与对策[J].中国蚕业, 2009(2): 20-23.
[18] MA Yuxin, NAVARRO B, ZHANG Zhixiang, et al. Identification and molecular characterization of a novel monopartite geminivirus associated with mulberry mosaic dwarf disease [J].The Journal of General Virology,2015,96(8):2421-2434.
[19] CHIUMENTI M, MORELLI M, DE STRADIS A, et al. Unusual genomic features of a badnavirus infecting mulberry [J]. The Journal of General Virology, 2016, 97(11): 3073-3087.
[20] LU Quanyou, WU Zujian, XIA Zhisong, et al. A new nepovirus identified in mulberry (Morus alba L.) in China [J]. Archives of Virology, 2015, 160(3): 851-855.