烟粉虱体内木尔坦棉花曲叶病毒的LAMP快速检测技术
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摘要
木尔坦棉花曲叶病毒(Cotton leaf curl Multan virus,CLCu Mu V)是引起世界范围内棉花曲叶病流行的主要病原之一,目前已入侵我国广东、广西、海南、福建、云南等地区。该病害由烟粉虱传播,随着烟粉虱扩散范围增加、危害不断加重,棉花曲叶病对我国棉花生产的潜在危害也日益增加。加强该病毒传播介体携带病毒的快速、特异性检测技术研发,对该病害的有效检疫与防控具有重要意义。本研究基于木尔坦棉花曲叶病毒(CLCu Mu V)的基因序列设计出LAMP检测4条特异性引物,建立LAMP扩增体系并优化扩增条件,扩增试验证明引物组合的特异性高,LAMP检测所需时间短,仅29 min即可定性检测CLCu Mu V扩增产物;该方法较普通PCR的扩增灵敏度高,可用于检测1头烟粉虱体内是否携带CLCu Mu V,简便易行,只通过颜色变绿或浊度变浑浊即可定性判断。建立的LAMP检测技术可被用于苗木上烟粉虱携带CLCu Mu V的早期检测,为介体昆虫带毒的检疫监测提供一种快速、准确和易操作的新技术。
Cotton leaf curl Multan virus( CLCu Mu V),transmitting by the silver leaf whitefly complex of Bemisia tabaci,has caused cotton curve leaf disease in the regions of Pakistan and India since 1997. The infested Hibiscus rose-sinensis and other host plants in the Hibisceae tribe of the family Malvaceae,were found in the region of South China. The disease was predicted to pose potential threat to cotton production in China once the whitefly complex as its vector disperse more widely. It is important and necessary to develop a quick,practicable tool for detecting CLCu Mu V presence in the whitefly body to efficiently prevent the virus from spread. Based on the conservative sequence of CLCu Mu V,4 primers specific to CLCu Mu V were designed and used in detection of the Begomovirus in a body of whitefly adult using Loop-Mediated Isothermal Amplification( LAMP). The reaction system for CLCu Mu V detection in the infested whitely body was set up and optimized. The LAMP technique was used to determine CLCu Mu V incident in a whitefly body within a short time of 29 min and the turbidity and green color could be oserved as indicators for CLCu Mu V present in the whitefly body. The consequences showed the tool is of specific and sensitive for CLCu Mu V detection in the whitefly.
Cotton leaf curl Multan virus( CLCu Mu V),transmitting by the silver leaf whitefly complex of Bemisia tabaci,has caused cotton curve leaf disease in the regions of Pakistan and India since 1997. The infested Hibiscus rose-sinensis and other host plants in the Hibisceae tribe of the family Malvaceae,were found in the region of South China. The disease was predicted to pose potential threat to cotton production in China once the whitefly complex as its vector disperse more widely. It is important and necessary to develop a quick,practicable tool for detecting CLCu Mu V presence in the whitefly body to efficiently prevent the virus from spread. Based on the conservative sequence of CLCu Mu V,4 primers specific to CLCu Mu V were designed and used in detection of the Begomovirus in a body of whitefly adult using Loop-Mediated Isothermal Amplification( LAMP). The reaction system for CLCu Mu V detection in the infested whitely body was set up and optimized. The LAMP technique was used to determine CLCu Mu V incident in a whitefly body within a short time of 29 min and the turbidity and green color could be oserved as indicators for CLCu Mu V present in the whitefly body. The consequences showed the tool is of specific and sensitive for CLCu Mu V detection in the whitefly.
引文
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Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Research,2000,28(12):e63.
Qin WT,Wang ZY,Zhang H.The progress of loop-mediated isothermal amplification(LAMP)and its application in detection of plant virus[J].Chinese Agricultural Science Bulletin,2013,29(21):170-174.[秦文韬,王中跃,张昊,环介导等.温扩增技术(LAMP)及其在植物病毒检测中的研究进展[J].中国农学通报,2013,29(21):170-174]
Rao YY,Shen LL,Zhu SF,et al.The economic losses of Cottone leaf curl virus invading China on cotton[J].Plant Quarantine,2011,25(3):12-16.[饶玉燕,沈玲丽,朱水芳,等.棉花曲叶病毒对棉花造成的经济损失评估[J].植物检疫,2011,25(3):12-16]
Sun M,Liang CZ,Gao HW,et al.Detection of CaM V 35S promoter by loop-mediated isothermal amplification[J].Food Science and Technology,2010,35(4):255-258.[孙敏,梁成珠,高宏伟,等.花椰菜花叶病毒CaM V35S启动子LAMP检测方法的建立[J].食品科技,2010,35(4):255-258]
Tang FF,Yang WK,Shao YL,et al.Prompt diagosis of Bombyx mori nucleopolyhedrosis virus by loop-mediated isothermal amplification[J].Chinese Journal of Applied Entomology,2013,50(4):1013-1019.[唐芬芬,杨伟克,邵榆岚,等.应用环介导等温扩增技术(LAMP)快速检测家蚕核型多角体病毒的研究[J].应用昆虫学报,2013,50(4):1013-1019]
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Tang YF,He ZF,Du ZG,et al.The complex of Cotton leaf curl Multan virus and its associated betasatellite molecule causing cotton leaf curl disease in Guangdong province[J].Scientia Agricultura Sinica。2015,48(16):3166-3175.[汤亚飞,何自福,杜振国,等.木尔坦棉花曲叶病毒及其伴随的β卫星分子复合侵染引起广东棉花曲叶病[J].中国农业科学,2015,48(16):3166-3175]
Wen WG,Yang CY,Cui JX,et al.Detection of Bean pod mottle virus by RT-LAMP[J].Plant Protection,2010,36(6):139-141.[闻伟刚,杨翠云,崔俊霞,等.RT-LAMP技术检测菜豆荚斑驳病毒的研究[J].植物保护,2010,36(6):139-141]
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Briddon RW.Cotton leaf curl disease,a multicomponent begomovirus complex[J].Molecular Plant Pathology,2003,4(6):427-434.
Cai JH,Xie K,Lin L,et al.Cotton leaf curl Multan virus newly reported to be associated with cotton leaf curl disease in China[J].Plant Pathology,2010,59(4):794-795.
Dinh DT,Le MT,Vuong CD,et al.An updated loop-mediated isothermal amplification method for rapid diagnosis of H5N1 avian influenza viruses[J].Trop Med.Health,2011.39(1):3-7.
Dong D,He ZF,Chai ZX.Detection of whitefly-transmitted geminivirus associated with the okra yellow vein leaf curl disease in Guangdong[J].Plant Protection,2010,36(1):65-68.[董迪,何自福,柴兆祥.广东黄秋葵黄脉曲叶病样中检测到烟粉虱传双生病毒[J].植物保护,2010,36(1):65-68]
Fukuta S,Lida T,Mizukami Y,et al.Detection of Japanese yam mosaic virus by RT-LAMP[J].Archives of Virology,2003,148:1713-1720.
Guo R.Cotton leaf curl virus disease will cause a serious threat to cotton production[J].China Plant Protection,2005,25(2):46-47.[郭荣.对棉花生产构成严重威胁的病害-棉花曲叶病毒病[J].中国植保导刊,2005,25(2):46-47]
Harrison BD,Liu YL,Khalid SH,et al.Detection and relationships of Cotton leaf curl virus and allied whitefly-transmitted geminiviruses occurring in Pakistan[J].Annals of Applied Biology,1997,130(1):61-75.
He ZF,Dong D,Li SF,et al.Threat of Cotton leaf curl Multan virus to China cotton production[J].Plant Protection,2010,36(2):147-149.[何自福,董迪,李世访,等.木尔坦棉花曲叶病毒已对我国棉花生产构成严重威胁[J].植物保护,2010,36(2):147-149]
He ZF,She XM,Tang YF.Cotton leaf curl Multan virus invading China and its damage potential[J].Journal of Biosafety,2012,21(2):87-92.[何自福,佘小漫,汤亚飞.入侵我国的木尔坦棉花曲叶病毒及其为害[J].生物安全学报,2012,21(2):87-92]
Kono T,Savan R,Sakai M,et al.Rapid detection of a fish iridovirus using loop-mediated isothermal amplification(LAMP)[J].Journal of Virological Methods,2004,121(2):155-161.
Liang XZ,Shi Y,Li JJ,et al.Detection of Cucurbit chlorotic yellows virus in the adult body the whitefly Bemisia tabaci using LAMP[J].Insect studies in Central China,2012,8:372.[梁相志,施艳,李静静,等.利用环介导等温扩增技术(LAMP)检测烟粉虱体内的瓜类褪绿黄化病毒(CCYV)[J].华中昆虫研究,2012,8:372]
Lin L,Cai JH,Luo EB,et al.Molecular identification and host range of a geminivirus infecting Hibiscus in Nanning city[J].Plant Protection,2011,37(4):44-47.[林林,蔡健和,罗恩波,等.南宁市朱槿曲叶病毒病病原分子鉴定和寄主范围研究[J].植物保护,2011,37(4):44-47]
Liu HY,Xin YY,Liu ZM,et al.Development of a RT-LAMP assay for rapid detection of potato virus A[J].Chinese Agricultural Science Bulletin,2015,31(11):143-147.[刘洪义,辛言言,刘忠梅,等.马铃薯A病毒RT-LAMP检测方法的建立[J].中国农学通报,2015,31(11):143-147]
Luo C,Zhang ZL.Research summary of Bemisia tabaci(Germadius)[J].Beijing Agricultural Science,2000,18(Suppl.):4-13.[罗晨,张芝利.烟粉虱Bemisia tabaci(Germadius)研究概述[J].北京农业科学,2000,18(增刊):4-13]
Mansoor S,Briddon RW,Bull SE,et al.Cotton leaf curl disease is associated with multiple monopartite begomoviruses supported by single DNAβ[J].Archives of Virology,2003,148(10):1969-1986.
Mao MJ,He ZF,Yu H,et al.Molecular characterization of Cotton leaf curl Multan virus and its satellite DNA that infects Hibiscus rosasinensis[J].Chinese Journal of Virology,2008,24(1):64-68.[毛明杰,何自福,虞皓,等.侵染朱槿的木尔坦棉花曲叶病毒及其卫星DNA全基因组结构特征[J].病毒学报,2008,24(1):64-68]
Nateshan HM,Muniyappa V,Swanson MM,et al.Host range,vector relations and serological relationships of Cotton leaf curl virus in south India[J].Annals of Applied Biology,1996,128(2):233-244.
Nie X Z.Reverse transcription loop-mediated isothermal amplification of DNA for detection of potato virus Y[J].Plant Disease,2005(89):605-610.
Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Research,2000,28(12):e63.
Qin WT,Wang ZY,Zhang H.The progress of loop-mediated isothermal amplification(LAMP)and its application in detection of plant virus[J].Chinese Agricultural Science Bulletin,2013,29(21):170-174.[秦文韬,王中跃,张昊,环介导等.温扩增技术(LAMP)及其在植物病毒检测中的研究进展[J].中国农学通报,2013,29(21):170-174]
Rao YY,Shen LL,Zhu SF,et al.The economic losses of Cottone leaf curl virus invading China on cotton[J].Plant Quarantine,2011,25(3):12-16.[饶玉燕,沈玲丽,朱水芳,等.棉花曲叶病毒对棉花造成的经济损失评估[J].植物检疫,2011,25(3):12-16]
Sun M,Liang CZ,Gao HW,et al.Detection of CaM V 35S promoter by loop-mediated isothermal amplification[J].Food Science and Technology,2010,35(4):255-258.[孙敏,梁成珠,高宏伟,等.花椰菜花叶病毒CaM V35S启动子LAMP检测方法的建立[J].食品科技,2010,35(4):255-258]
Tang FF,Yang WK,Shao YL,et al.Prompt diagosis of Bombyx mori nucleopolyhedrosis virus by loop-mediated isothermal amplification[J].Chinese Journal of Applied Entomology,2013,50(4):1013-1019.[唐芬芬,杨伟克,邵榆岚,等.应用环介导等温扩增技术(LAMP)快速检测家蚕核型多角体病毒的研究[J].应用昆虫学报,2013,50(4):1013-1019]
Tang YF,He ZF,Du ZG,et al.Molecular characterization of the Cotton leaf curl Multan virus infecting Malvaiscus arboreus[J].Acta Phytopathologiga Sinica,2013,43(2):120-127.[汤亚飞,何自福,杜振国等.侵染垂花悬铃花的木尔坦棉花曲叶病毒分子特征研究[J].植物病理学,2013,43(2):120-127]
Tang YF,He ZF,Du ZG,et al.The complex of Cotton leaf curl Multan virus and its associated betasatellite molecule causing cotton leaf curl disease in Guangdong province[J].Scientia Agricultura Sinica。2015,48(16):3166-3175.[汤亚飞,何自福,杜振国,等.木尔坦棉花曲叶病毒及其伴随的β卫星分子复合侵染引起广东棉花曲叶病[J].中国农业科学,2015,48(16):3166-3175]
Wen WG,Yang CY,Cui JX,et al.Detection of Bean pod mottle virus by RT-LAMP[J].Plant Protection,2010,36(6):139-141.[闻伟刚,杨翠云,崔俊霞,等.RT-LAMP技术检测菜豆荚斑驳病毒的研究[J].植物保护,2010,36(6):139-141]
Yuan DW,Liu JS,Guo DC et al.Application of a loop-mediated isothermal amplification method in detection of RHDV[J].Chinese Veterinary Science,2013,43(4):390-395.[原冬伟,刘家森,郭东春,等.环介导等温扩增技术兔出血症病毒检测中的应用[J].中国兽医科学,2013,43(4):390-395]
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