重组酶聚合酶扩增技术在番茄黄化曲叶病毒检测中的应用
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摘要
番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)是引起番茄黄化曲叶病毒病的病原,在生产中造成严重危害,开发快速准确的检测方法对该病害的防控具有重要意义。依据番茄黄化曲叶病毒DNA-A基因序列,设计用于重组酶聚合酶等温扩增(RPA)检测的特异性引物,建立了TYLCV的重组酶聚合酶等温扩增检测方法,并分析该方法的特异性和灵敏度。结果表明,建立的TYLCV-RPA方法可从TYLCV阳性的番茄样品中扩增出343 bp的特异性条带,反应条件为37℃恒温下40 min,扩增时间短,对设备要求低,且与番茄其他病毒无交叉反应,特异性好,灵敏度可达到PCR方法的10倍,适用于TYLCV的快速检测。
Tomato yellow leaf curl virus(TYLCV)is an etiology of Tomato yellow leaf curl disease(TYLCD),which will cause serious damages to tomato production.It is of important significance to exploit rapid and accurate detection method for controlling this disease.According to DNA-A gene sequence of TYLCV,this study designed special RPA primers and established a method named TYLCV-RPA,and analyzed the specificity and sensitivity of this method.The results indicated that TYLCV-RPA method could amplify specific band of 343 bp from sample tomatoes with positive TYLCV.The reaction condition was 37 ℃ for 40 min.This method needed short time period for amplification with low requirement for equipment.It had no cross reaction with the other viruses and had good specificity.Its sensitivity was 10 times of the PCR method.It is suitable for rapid detection of TYLCV.
Tomato yellow leaf curl virus(TYLCV)is an etiology of Tomato yellow leaf curl disease(TYLCD),which will cause serious damages to tomato production.It is of important significance to exploit rapid and accurate detection method for controlling this disease.According to DNA-A gene sequence of TYLCV,this study designed special RPA primers and established a method named TYLCV-RPA,and analyzed the specificity and sensitivity of this method.The results indicated that TYLCV-RPA method could amplify specific band of 343 bp from sample tomatoes with positive TYLCV.The reaction condition was 37 ℃ for 40 min.This method needed short time period for amplification with low requirement for equipment.It had no cross reaction with the other viruses and had good specificity.Its sensitivity was 10 times of the PCR method.It is suitable for rapid detection of TYLCV.
引文
邓婷婷,黄文胜,程奇,李新实,陈颖.2015.重组酶聚合酶扩增技术检测转基因水稻中的Cry1Ab/c基因.中国食品学报,15(3):187-193.
樊晓旭,赵永刚,李林,吴晓东,王志亮.2016.重组酶聚合酶扩增技术在疾病快速检测中的研究进展.中国动物检疫,33(8):72-77.
季英华,周晓伟,张晖,周益军.2013,应用DIBA快速检测番茄黄化曲叶病毒.江苏农业学报,29(3):685-687.
金凤媚,薛俊,郏艳红,周祥明,王建江,刘仲齐.2011.天津地区番茄黄化曲叶病毒DNA-A的克隆和序列分析.华北农学报,26(1):58-62.
李常保,崔彦玲,张丽英,李传友.2012.番茄黄化曲叶病毒的快速分子检测.遗传,34(3):366-370.
李明远.2009.黄化曲叶病毒病传入北京,番茄生产面临威胁.植物保护,35(6):179-180.
刘静,武国干,吴潇,刘华,王金斌,吕贝贝,蒋玮,唐雪明.2018.重组酶聚合酶扩增技术快速检测转基因玉米Bt11.上海农业学报,34(1):20-24.
秦文韬,王忠跃,张昊.2013.环介导恒温扩增技术(lamp)及其在植物病毒检测中的研究进展.中国农学通报,(21):170-174.
宋晰,师迎春,张世晨,梁彦,王廿,陈善义,周涛.2013.北京地区番茄黄化曲叶病病毒分离物测定及株系的初步鉴定.植物病理学报,43(2):113-119.
汪琳,罗英,周琦,赖平安,柏亚铎.2011.核酸恒温扩增技术研究进展.生物技术通讯,22(2):296-302.
魏梅生,田茜,赵文军,李桂芬,孔君.2016.番茄细菌性叶斑病菌RPA检测技术.植物保护,42(1):150-153.
吴耀东,徐民俊,郑文斌,冯胜勇,朱兴全.2016.重组酶聚合酶扩增技术及其在动物病原快速检测中的应用.中国兽医学报,36(10):1797-1802.
熊艳,杨帅,青玲,周常勇,孙现超,杨水英.2011.四川番茄黄化曲叶病病原分子鉴定及变异分析.中国农业科学,44(3):477-484.
徐潮,李亮,金芜军,宛煜嵩.2014.荧光RPA技术检测转基因水稻科丰6号.分子植物育种,12(5):875-880.
余玲玲,钱亚娟,周雪平.2008.用滚环扩增技术检测双生病毒.植物病理学报,38(6):570-575.
余文贵,赵统敏,杨玛丽,赵丽萍,季英华,周益军.2009.山东、安徽两省栽培番茄烟粉虱传双生病毒的PCR检测及序列分析.江苏农业学报,25(4):747-751.
张娜,乾义柯,魏霜,胡白石,陆平,赛铁尔汗,刘中勇,张永江,张祥林.2016.基于重组酶聚合酶扩增技术(RPA)的葡萄卷叶伴随病毒3号检测方法.新疆农业科学,53(2):302-308.
张伟,徐硕,陈德鑫.2013.常用植物病毒病检测技术比较.南方农业,7(12):24-28.
赵统敏,余文贵,周益军,季英华.2007.江苏省番茄黄化曲叶病毒病(TYLCD)的发生与诊断初报.江苏农业学报,23(6):654-655.
周莹,李兴红,刘建华,姜京宇,王曙峰,燕继晔.2010.河北省番茄黄化曲叶病毒病的分子鉴定初报.植物保护,36(1):60-64.
周莹,尚巧霞,罗晨,乔广行,刘梅,张玮,燕继晔.2016.番茄黄化曲叶病毒的鉴定与群体进化分析.植物保护,42(1):106-111.
Cohen S,Harpaz I.1964.Periodic,rather than continual acquisition of a new tomato virus by its vector,the tobacco whitefly(Bemisia tabaci Gennadius).Entomologia Experimentalis Et Applicata,7(2):155-166.
Moffat A S.1999.Geminiviruses emerge as serious crop threat.Science,286(5446):1835.
Moriones E,Navas-Castillo J.2000.Tomato yellow leaf curl virus,an emerging virus complex causing epidemics worldwide.Virus Research,71(2):123-134.
Sondra G,Lazarowitz R J.1992.Geminiviruses:genome structure and gene function.Critical Reviews in Plant Sciences,11(4):327-349.
Varsani A,Navas-Castillo J,Moriones E,Hernández-Zepeda C,Idris A,Brown J K,Zerbini F M.2014.Establishment of three new genera in the family Geminiviridae:Becurtovirus,Eragrovirus and Turncurtovirus.Archives of Virology,159:2193-2203.
Wu J B,Dai F M,Zhou X P.2006.First report of Tomato yellow leaf curl virus in China.Plant Disease,90(10):1359.
樊晓旭,赵永刚,李林,吴晓东,王志亮.2016.重组酶聚合酶扩增技术在疾病快速检测中的研究进展.中国动物检疫,33(8):72-77.
季英华,周晓伟,张晖,周益军.2013,应用DIBA快速检测番茄黄化曲叶病毒.江苏农业学报,29(3):685-687.
金凤媚,薛俊,郏艳红,周祥明,王建江,刘仲齐.2011.天津地区番茄黄化曲叶病毒DNA-A的克隆和序列分析.华北农学报,26(1):58-62.
李常保,崔彦玲,张丽英,李传友.2012.番茄黄化曲叶病毒的快速分子检测.遗传,34(3):366-370.
李明远.2009.黄化曲叶病毒病传入北京,番茄生产面临威胁.植物保护,35(6):179-180.
刘静,武国干,吴潇,刘华,王金斌,吕贝贝,蒋玮,唐雪明.2018.重组酶聚合酶扩增技术快速检测转基因玉米Bt11.上海农业学报,34(1):20-24.
秦文韬,王忠跃,张昊.2013.环介导恒温扩增技术(lamp)及其在植物病毒检测中的研究进展.中国农学通报,(21):170-174.
宋晰,师迎春,张世晨,梁彦,王廿,陈善义,周涛.2013.北京地区番茄黄化曲叶病病毒分离物测定及株系的初步鉴定.植物病理学报,43(2):113-119.
汪琳,罗英,周琦,赖平安,柏亚铎.2011.核酸恒温扩增技术研究进展.生物技术通讯,22(2):296-302.
魏梅生,田茜,赵文军,李桂芬,孔君.2016.番茄细菌性叶斑病菌RPA检测技术.植物保护,42(1):150-153.
吴耀东,徐民俊,郑文斌,冯胜勇,朱兴全.2016.重组酶聚合酶扩增技术及其在动物病原快速检测中的应用.中国兽医学报,36(10):1797-1802.
熊艳,杨帅,青玲,周常勇,孙现超,杨水英.2011.四川番茄黄化曲叶病病原分子鉴定及变异分析.中国农业科学,44(3):477-484.
徐潮,李亮,金芜军,宛煜嵩.2014.荧光RPA技术检测转基因水稻科丰6号.分子植物育种,12(5):875-880.
余玲玲,钱亚娟,周雪平.2008.用滚环扩增技术检测双生病毒.植物病理学报,38(6):570-575.
余文贵,赵统敏,杨玛丽,赵丽萍,季英华,周益军.2009.山东、安徽两省栽培番茄烟粉虱传双生病毒的PCR检测及序列分析.江苏农业学报,25(4):747-751.
张娜,乾义柯,魏霜,胡白石,陆平,赛铁尔汗,刘中勇,张永江,张祥林.2016.基于重组酶聚合酶扩增技术(RPA)的葡萄卷叶伴随病毒3号检测方法.新疆农业科学,53(2):302-308.
张伟,徐硕,陈德鑫.2013.常用植物病毒病检测技术比较.南方农业,7(12):24-28.
赵统敏,余文贵,周益军,季英华.2007.江苏省番茄黄化曲叶病毒病(TYLCD)的发生与诊断初报.江苏农业学报,23(6):654-655.
周莹,李兴红,刘建华,姜京宇,王曙峰,燕继晔.2010.河北省番茄黄化曲叶病毒病的分子鉴定初报.植物保护,36(1):60-64.
周莹,尚巧霞,罗晨,乔广行,刘梅,张玮,燕继晔.2016.番茄黄化曲叶病毒的鉴定与群体进化分析.植物保护,42(1):106-111.
Cohen S,Harpaz I.1964.Periodic,rather than continual acquisition of a new tomato virus by its vector,the tobacco whitefly(Bemisia tabaci Gennadius).Entomologia Experimentalis Et Applicata,7(2):155-166.
Moffat A S.1999.Geminiviruses emerge as serious crop threat.Science,286(5446):1835.
Moriones E,Navas-Castillo J.2000.Tomato yellow leaf curl virus,an emerging virus complex causing epidemics worldwide.Virus Research,71(2):123-134.
Sondra G,Lazarowitz R J.1992.Geminiviruses:genome structure and gene function.Critical Reviews in Plant Sciences,11(4):327-349.
Varsani A,Navas-Castillo J,Moriones E,Hernández-Zepeda C,Idris A,Brown J K,Zerbini F M.2014.Establishment of three new genera in the family Geminiviridae:Becurtovirus,Eragrovirus and Turncurtovirus.Archives of Virology,159:2193-2203.
Wu J B,Dai F M,Zhou X P.2006.First report of Tomato yellow leaf curl virus in China.Plant Disease,90(10):1359.