柑橘鳞皮病毒RT-LAMP检测方法的建立与应用
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摘要
根据NCBI数据库柑橘鳞皮病毒(Citrus psorosis virus,CPsV)的外壳蛋白(coat protein,CP)基因序列设计了4组特异性引物,通过一系列优化,筛选并获得1组特异性引物。以感染CPsV的柑橘叶片总RNA为模板,采用一步法RT-LAMP(One-step reverse transcription loop-mediated isothermal amplification),在60℃下反应60 min,对扩增产物进行琼脂糖电泳分析,同时测定其特异性和灵敏度。结果表明,建立的RT-LAMP检测方法能特异性扩增CPsV,灵敏度为RT-PCR的10倍。用RT-LAMP方法对87个田间样品进行检测,发现有5个样品感染了CPsV,其检测结果与RT-PCR法一致。RT-LAMP法具有特异性强、灵敏度高、操作简单、快速等特点,适合对CPsV样品的快速检测与鉴定。
Four sets of specific primer pairs in the conserved region of the CP(coat protein)gene of Citrus psorosis virus(CPsV)were designed for One-step RT-LAMP detection system based on NCBI database. One feasible pair of primers was screened to be suitable for this system. Template RNA extracted from CPsV infected citrus leaves was used for One-step RT-LAMP,and isothermal conditions were optimized to be 60 ℃ for 60 minutes. The agarose gel electrophoresis analysis of RT-LAMP products indicated that the established RT-LAMP system was specific and sensitive in detecting CPsV and 10-fold higher than RT-PCR method in sensitivity. Of 87 samples detected by this RT-LAMP system,five were found to be infected by CPsV,showing the detection result was consistent with RT-PCR method. Hence this RT-LAMP system is applicable for rapid and accurate detection and identification of CPsV.
Four sets of specific primer pairs in the conserved region of the CP(coat protein)gene of Citrus psorosis virus(CPsV)were designed for One-step RT-LAMP detection system based on NCBI database. One feasible pair of primers was screened to be suitable for this system. Template RNA extracted from CPsV infected citrus leaves was used for One-step RT-LAMP,and isothermal conditions were optimized to be 60 ℃ for 60 minutes. The agarose gel electrophoresis analysis of RT-LAMP products indicated that the established RT-LAMP system was specific and sensitive in detecting CPsV and 10-fold higher than RT-PCR method in sensitivity. Of 87 samples detected by this RT-LAMP system,five were found to be infected by CPsV,showing the detection result was consistent with RT-PCR method. Hence this RT-LAMP system is applicable for rapid and accurate detection and identification of CPsV.
引文
Achachi A,Ait Barka E,Ibriz M.2014.Recent advances in Citrus psorosis virus.Virus Disease,25(3):261-276
Achachi A,Jijakli M H,Fahime E E,Soulaymani A,Ibriz M.2015.Detection of Citrus psorosis virus, using an improved one-step RT-PCR.Arabian Journal for Science&Engineering,40(1):7-13
Barthe G A,Ceccardi T L,Manj unath K L,Derrick K S.1998.Citrus psorosis virus:nucleotide sequencing of the coat protein gene and detection by hybridization and RT-PCR.The Journal of General Virology,79(6):1531-1537
Chen Guo-qing,Wang Hong-xiang,Yan Sen-xiang.1996.The occurrence and biochemical identification of citrus ringspot disease.Zhejiang Citrus13(3):43.(in Chinese)陈国庆,王洪祥,严森祥.1996.柑桔环斑病的发生与生物学鉴定初报.浙江柑橘,13(3):43.
Duan Shuo.2013.Establishment of reverse transcription loop-mediated isothermal amplification for detecting Citrus tatter leaf virus and its prokaryotic expression of coat protein gene[M.D.Dissertation].Chongqing:Southwest University.(in Chinese)段硕,2013.柑橘碎叶病毒RT-LAMP检测体系建立及其外壳蛋白基因的原核表达[硕士论文].重庆:西南大学.
Garcia M L,Bo E D,a Graca J V,Gago-Zachert S,Hammond J,Moreno P,Natsuaki T,Pallas V,Navarro J A,Reyes C A,Luna G R,Sasaya T,Tzannetakis I E,Vaira A M,Verbeek M.2017.ICTV virus taxonomy profile:Ophioviridae.Journal of General Virology,98:1161-1162.
Huang Li,Su Hua-nan,Tang Ke-zhi,Huang Ai-jun,Zhou Chang-yong,Li Zhong-an.2012.Establishment and application of loop-mediated isothermal amplification assay for the detection of Citrus Huanglongbing.Journal of Fruit Science,29(6):1121-1126.(in Chinese)黄丽,苏华楠,唐科志,黄爱军,周常勇,李中安.2012.柑橘黄龙病LAMP快速检测方法的建立及应用.果树学报,29(6):1121-1126.
Jiang Shan-shan,Feng Jia,Zhang Mei,Wang Sheng-ji,Xin Zhi-mei,Wu Bin,Xin Xiang-qi.2018.Development of RT-LAMP assay for rapid detection of Sweet potato feathery mottle virus(SPFMV).Scientia Agricultura Sinica,51(7):1294-1302.(in Chinese)姜珊珊,冯佳,张眉,王升吉,辛志梅,吴斌,辛相启.2018.甘薯羽状斑驳病毒RT-LAMP快速检测方法的建立.中国农业科学,51(7):1294-1302.
Li Huawei,Xu Yongqing,Luo Wenbin,Ji Rongchang,Liu Zhonghua,Xu Guo-chun,Zhang Hong,Li Guoliang,Lin Zhaomiao,Qiu Yong-xiang,Qiu Sixin,Tang Hao.2018.Establishment of loop-mediated isothermal amplification assay for detection of Potato virus S drdinary strain.Acta Horticulturae Sinica,45(8):1613-1620.(in Chinese)李华伟,许泳清,罗文彬,纪荣昌,刘中华,许国春,张鸿,李国良,林赵淼,邱永祥,邱思鑫,汤浩.2018,马铃薯S病毒PVS~o株系RT-LAMP检测方法的建立及应用.园艺学报,45(8):1613-1620.
Li Zhanbiao,Xie Huiting,Cui Lixian,Su Qin, Qin Bixia,Yang Shian,Deng Tiejun,Cai Jianhe.2017.Establishment of reverse-transcription loop-mediated isothermal amplication assay for rapid detection of Melon yellow spot virus.Acta Horticulturae Sinica,44(5):1005-1010.(in Chinese)李战彪,谢慧婷,崔丽贤,苏琴,秦碧霞,杨世安,邓铁军,蔡健和.2017.甜瓜黄斑病毒(MYSV)RT-LAMP快速检测方法的建立.园艺学报,44(5):1005-1010.
Liu Kehong,Chen Hongming,Zhou Yan,Li Zhongan.2015.Establishment of RT-LAMP assay for detection of Citrus yellow vein clearing virus.Acta Horticulturae Sinica,42(5):997-1002.(in Chinese)刘科宏,陈洪明,周彦,李中安.2015.柑橘黄化脉明病毒RT-LAMP检测方法的建立.园艺学报,42(5):997-1002.
Liu Kehong,Chen Hongming,Zhou Yan,Song Zhen,Li Zhongan.2017.Establishment of LAMP assay for detection of Citrus chlorotic dwarf-associated virus(CCDaV).Acta Horticulturae Sinica,44(5):999-1004.(in Chinese)刘科宏,陈洪明,周彦,宋震,李中安.2017.柑橘褪绿矮化相关病毒LAMP检测体系的建立.园艺学报,44(5):999-1004.
Lu Yongcan,Yao Bingyu,Wang Guoping,Hong Ni.2018.The detection of ACLSV and ASPV in pear plants by RT-LAMP assays.Journal of Virological Methods,252:80-85.
Martin S,Alioto D,Milne R G, Guerri J,Moreno P.2002.Detection of Citrus psorosis virus in field trees by direct tissue blot immunoassay in comparison with ELISA,symptomatology,biological indexing and cross-protection tests.Plant Pathology,51(2):134-141.
Martin S,Alioto D,Milne R G, Garnsey S M,Garcia M L,Grau O,Guerri J,Moreno P.2004.Detection of Citrus psorosis virus, by ELISA,molecular hybridization,RT-PCR and immunosorbent electron microscopy and its association with citrus psorosis disease.European Journal of Plant Pathology,110(7):747-757.
Naum-Ongania G,Gago-Zachert S,Pena E,Grau O,Garcia M L.2003.Citrus psorosis virus RNA 1 is of negative polarity and potentially encodesin its complementary strand a 24K protein of unknown function and 280K putative RNA dependent RNA polymerase.Virus Research,96(1):49-61.
Notomi T,Okayama H,Masubuchi H,Yonekawa T,Watanabe K,Amino N,Hase T.2000.Loop-mediated isothermal amplification of DNA.Nucleic Acids Research,28(12):E63.
Palle S R,Miao H,Seyran M,Louzada E S,Graca J V D,Skaria M.2005.Evidence for association of Citrus psorosis virus with symptomatic trees and an Olpidium-like fungus in Texas.International Organization of Citrus Virologists Conference Proceedings(1957-2010),16:423-426.
Robles Luna G,Pena E J,Bomiego M B,Heinlein M,Garcia M L.2013.Ophioviruses CPsV and MiLBVV movement protein is encoded in RNA2 and interacts with the coat protein.Virology,441(2):152-161.
Roistacher C N.1993.Psorosis-a review//Moreno P,da Graca J V,Timmer L W.Proceedings of 12th conference on international organisation of citrus virologists.Riverside:University of California:139-154.
Vaira A M,Hansen M A,Murphy C,Reinsel M D,Hammond J.2009.First report of Freesia sneak virus in Freesia sp.in Virginia.Plant Disease,93(9):965.
Vaira A M,Kleynhans R,Hammond J.2007.First report of Freesia sneak virus infecting Lachenalia cultivars in South Africa.Plant Disease,91(6):770.
Warghane A,Misra P,Bhos S,Biswas K K,Sharma A K,Reddy M K,Ghosh D K.2017.Development of a simple and rapid reverse transcription-loop mediated isothermal amplification(RT-LAMP)assay for sensitive detection of Citrus tristeza virus.Journal of Virological Methods,250:6-10.
Zhang Lun,Yin You-ping,Wu Yu-jia,Wang Zhong-kang.2013.Regular LAMP and fast LAMP for the detection ofXanthomonas axon,opodis pv.citri.Plant Protection,39(3):95-101.(in Chinese)张仑,殷幼平,吴瑜佳,王中康.2013.柑橘溃疡病菌的普通LAMP及快速LAMP检测方法的建立.植物保护,39(3):95-101.
Zhang Shuang-na,Li Zheng-nan,Fan Xu-dong,Zhang Zun-ping,Ren Fang,Hu Guo-jun,Dong Ya-feng.2018.Establishment of RT-LAMP assay for detection of Apple chlorotic leaf spot virus(ACLSV).Scientia Agricultura Sinica,51(9):1706-1716.(in Chinese)张双纳,李正男,范旭东,张尊平,任芳,胡国君,董雅凤.2018.苹果褪绿叶斑病毒RT-LAMP检测方法的建立.中国农业科学,51(9):1706-1716.
Achachi A,Jijakli M H,Fahime E E,Soulaymani A,Ibriz M.2015.Detection of Citrus psorosis virus, using an improved one-step RT-PCR.Arabian Journal for Science&Engineering,40(1):7-13
Barthe G A,Ceccardi T L,Manj unath K L,Derrick K S.1998.Citrus psorosis virus:nucleotide sequencing of the coat protein gene and detection by hybridization and RT-PCR.The Journal of General Virology,79(6):1531-1537
Chen Guo-qing,Wang Hong-xiang,Yan Sen-xiang.1996.The occurrence and biochemical identification of citrus ringspot disease.Zhejiang Citrus13(3):43.(in Chinese)陈国庆,王洪祥,严森祥.1996.柑桔环斑病的发生与生物学鉴定初报.浙江柑橘,13(3):43.
Duan Shuo.2013.Establishment of reverse transcription loop-mediated isothermal amplification for detecting Citrus tatter leaf virus and its prokaryotic expression of coat protein gene[M.D.Dissertation].Chongqing:Southwest University.(in Chinese)段硕,2013.柑橘碎叶病毒RT-LAMP检测体系建立及其外壳蛋白基因的原核表达[硕士论文].重庆:西南大学.
Garcia M L,Bo E D,a Graca J V,Gago-Zachert S,Hammond J,Moreno P,Natsuaki T,Pallas V,Navarro J A,Reyes C A,Luna G R,Sasaya T,Tzannetakis I E,Vaira A M,Verbeek M.2017.ICTV virus taxonomy profile:Ophioviridae.Journal of General Virology,98:1161-1162.
Huang Li,Su Hua-nan,Tang Ke-zhi,Huang Ai-jun,Zhou Chang-yong,Li Zhong-an.2012.Establishment and application of loop-mediated isothermal amplification assay for the detection of Citrus Huanglongbing.Journal of Fruit Science,29(6):1121-1126.(in Chinese)黄丽,苏华楠,唐科志,黄爱军,周常勇,李中安.2012.柑橘黄龙病LAMP快速检测方法的建立及应用.果树学报,29(6):1121-1126.
Jiang Shan-shan,Feng Jia,Zhang Mei,Wang Sheng-ji,Xin Zhi-mei,Wu Bin,Xin Xiang-qi.2018.Development of RT-LAMP assay for rapid detection of Sweet potato feathery mottle virus(SPFMV).Scientia Agricultura Sinica,51(7):1294-1302.(in Chinese)姜珊珊,冯佳,张眉,王升吉,辛志梅,吴斌,辛相启.2018.甘薯羽状斑驳病毒RT-LAMP快速检测方法的建立.中国农业科学,51(7):1294-1302.
Li Huawei,Xu Yongqing,Luo Wenbin,Ji Rongchang,Liu Zhonghua,Xu Guo-chun,Zhang Hong,Li Guoliang,Lin Zhaomiao,Qiu Yong-xiang,Qiu Sixin,Tang Hao.2018.Establishment of loop-mediated isothermal amplification assay for detection of Potato virus S drdinary strain.Acta Horticulturae Sinica,45(8):1613-1620.(in Chinese)李华伟,许泳清,罗文彬,纪荣昌,刘中华,许国春,张鸿,李国良,林赵淼,邱永祥,邱思鑫,汤浩.2018,马铃薯S病毒PVS~o株系RT-LAMP检测方法的建立及应用.园艺学报,45(8):1613-1620.
Li Zhanbiao,Xie Huiting,Cui Lixian,Su Qin, Qin Bixia,Yang Shian,Deng Tiejun,Cai Jianhe.2017.Establishment of reverse-transcription loop-mediated isothermal amplication assay for rapid detection of Melon yellow spot virus.Acta Horticulturae Sinica,44(5):1005-1010.(in Chinese)李战彪,谢慧婷,崔丽贤,苏琴,秦碧霞,杨世安,邓铁军,蔡健和.2017.甜瓜黄斑病毒(MYSV)RT-LAMP快速检测方法的建立.园艺学报,44(5):1005-1010.
Liu Kehong,Chen Hongming,Zhou Yan,Li Zhongan.2015.Establishment of RT-LAMP assay for detection of Citrus yellow vein clearing virus.Acta Horticulturae Sinica,42(5):997-1002.(in Chinese)刘科宏,陈洪明,周彦,李中安.2015.柑橘黄化脉明病毒RT-LAMP检测方法的建立.园艺学报,42(5):997-1002.
Liu Kehong,Chen Hongming,Zhou Yan,Song Zhen,Li Zhongan.2017.Establishment of LAMP assay for detection of Citrus chlorotic dwarf-associated virus(CCDaV).Acta Horticulturae Sinica,44(5):999-1004.(in Chinese)刘科宏,陈洪明,周彦,宋震,李中安.2017.柑橘褪绿矮化相关病毒LAMP检测体系的建立.园艺学报,44(5):999-1004.
Lu Yongcan,Yao Bingyu,Wang Guoping,Hong Ni.2018.The detection of ACLSV and ASPV in pear plants by RT-LAMP assays.Journal of Virological Methods,252:80-85.
Martin S,Alioto D,Milne R G, Guerri J,Moreno P.2002.Detection of Citrus psorosis virus in field trees by direct tissue blot immunoassay in comparison with ELISA,symptomatology,biological indexing and cross-protection tests.Plant Pathology,51(2):134-141.
Martin S,Alioto D,Milne R G, Garnsey S M,Garcia M L,Grau O,Guerri J,Moreno P.2004.Detection of Citrus psorosis virus, by ELISA,molecular hybridization,RT-PCR and immunosorbent electron microscopy and its association with citrus psorosis disease.European Journal of Plant Pathology,110(7):747-757.
Naum-Ongania G,Gago-Zachert S,Pena E,Grau O,Garcia M L.2003.Citrus psorosis virus RNA 1 is of negative polarity and potentially encodesin its complementary strand a 24K protein of unknown function and 280K putative RNA dependent RNA polymerase.Virus Research,96(1):49-61.
Notomi T,Okayama H,Masubuchi H,Yonekawa T,Watanabe K,Amino N,Hase T.2000.Loop-mediated isothermal amplification of DNA.Nucleic Acids Research,28(12):E63.
Palle S R,Miao H,Seyran M,Louzada E S,Graca J V D,Skaria M.2005.Evidence for association of Citrus psorosis virus with symptomatic trees and an Olpidium-like fungus in Texas.International Organization of Citrus Virologists Conference Proceedings(1957-2010),16:423-426.
Robles Luna G,Pena E J,Bomiego M B,Heinlein M,Garcia M L.2013.Ophioviruses CPsV and MiLBVV movement protein is encoded in RNA2 and interacts with the coat protein.Virology,441(2):152-161.
Roistacher C N.1993.Psorosis-a review//Moreno P,da Graca J V,Timmer L W.Proceedings of 12th conference on international organisation of citrus virologists.Riverside:University of California:139-154.
Vaira A M,Hansen M A,Murphy C,Reinsel M D,Hammond J.2009.First report of Freesia sneak virus in Freesia sp.in Virginia.Plant Disease,93(9):965.
Vaira A M,Kleynhans R,Hammond J.2007.First report of Freesia sneak virus infecting Lachenalia cultivars in South Africa.Plant Disease,91(6):770.
Warghane A,Misra P,Bhos S,Biswas K K,Sharma A K,Reddy M K,Ghosh D K.2017.Development of a simple and rapid reverse transcription-loop mediated isothermal amplification(RT-LAMP)assay for sensitive detection of Citrus tristeza virus.Journal of Virological Methods,250:6-10.
Zhang Lun,Yin You-ping,Wu Yu-jia,Wang Zhong-kang.2013.Regular LAMP and fast LAMP for the detection ofXanthomonas axon,opodis pv.citri.Plant Protection,39(3):95-101.(in Chinese)张仑,殷幼平,吴瑜佳,王中康.2013.柑橘溃疡病菌的普通LAMP及快速LAMP检测方法的建立.植物保护,39(3):95-101.
Zhang Shuang-na,Li Zheng-nan,Fan Xu-dong,Zhang Zun-ping,Ren Fang,Hu Guo-jun,Dong Ya-feng.2018.Establishment of RT-LAMP assay for detection of Apple chlorotic leaf spot virus(ACLSV).Scientia Agricultura Sinica,51(9):1706-1716.(in Chinese)张双纳,李正男,范旭东,张尊平,任芳,胡国君,董雅凤.2018.苹果褪绿叶斑病毒RT-LAMP检测方法的建立.中国农业科学,51(9):1706-1716.