利用环介导等温扩增方法快速检测瓜类细菌性果斑病菌和番茄细菌性溃疡病菌
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摘要
为建立简单、快速和灵敏地检测瓜类细菌性果斑病菌Acidovorax avenae subsp. citrulli(Aac)和番茄细菌性溃疡病菌Clavibacter michiganensis subsp. michiganensis(Cmm)的环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法,以Aac的ugpB基因和Cmm的micA基因为靶标,分别设计、合成和筛选特异性引物,摸索和优化各项反应条件和反应体系,成功建立了以钙黄绿素颜色为指示且只需要金属浴恒温反应30～60 min的LAMP扩增体系。特异性分析表明该LAMP方法可以快速检出5株不同的Aac菌株和2株不同的Cmm菌株,其它对照菌株如燕麦嗜酸菌燕麦亚种A. avenae subsp. avenae、丁香假单胞菌Pseudomonas syringae、水稻黄单胞菌水稻致病变种Xanthomonas oryzae pv. oryzae和茄科雷尔氏菌Ralstonia solanacearum则呈现阴性反应;引物比目前报道的LAMP引物有更高的DNA样品检测灵敏度,Aac和Cmm的灵敏度分别为1.72×10~2fg/μL和1.26×10~2fg/μL。研究结果表明,所设计的Aac和Cmm引物特异性好、灵敏度高,更有利于从源头上控制这2种检疫性细菌病害的流行和传播。
To establish a simple, sensitive and rapid loop-mediated isothermal amplification(LAMP)method for the detection of Acidovorax avenae subsp. citrulli(Aac) causing melon bacterial fruit blotch and Clavibacter michiganensis subsp. michiganensis(Cmm) causing tomato bacterial canker disease,ugpB gene of Aac and micA gene of Cmm were used as the target for design of LAMP primers. Following primers screening and optimization of reaction systems and conditions, the LAMP method was finally established based on 30-60 min incubation by using calcein as an indicator. It showed a good prospect for application due to the reduction in both the reaction time and cost. The results of specificity analysis indicated a positive reaction for the five strains of Aac and two strains of Cmm and a negative reaction for the reference strains A. avenae subsp. avenae, Pseudomonas syringae, Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, etc. In addition, the sensitivity of LAMP was 1.72×10~2 fg/μL for Aacand 1.26×10~2 fg/μL for Cmm, respectively, which were higher than those of the previous reports. In general, the LAMP system is highly sensitive and specific and helpful for control of the epidemics and spread of the two bacterial diseases.
To establish a simple, sensitive and rapid loop-mediated isothermal amplification(LAMP)method for the detection of Acidovorax avenae subsp. citrulli(Aac) causing melon bacterial fruit blotch and Clavibacter michiganensis subsp. michiganensis(Cmm) causing tomato bacterial canker disease,ugpB gene of Aac and micA gene of Cmm were used as the target for design of LAMP primers. Following primers screening and optimization of reaction systems and conditions, the LAMP method was finally established based on 30-60 min incubation by using calcein as an indicator. It showed a good prospect for application due to the reduction in both the reaction time and cost. The results of specificity analysis indicated a positive reaction for the five strains of Aac and two strains of Cmm and a negative reaction for the reference strains A. avenae subsp. avenae, Pseudomonas syringae, Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, etc. In addition, the sensitivity of LAMP was 1.72×10~2 fg/μL for Aacand 1.26×10~2 fg/μL for Cmm, respectively, which were higher than those of the previous reports. In general, the LAMP system is highly sensitive and specific and helpful for control of the epidemics and spread of the two bacterial diseases.
引文
Arimatsu Y,Kaewkes S,Laha T,Sripa B.2015.Specific diagnosis of Opisthorchis viverrini using loop-mediated isothermal amplification(LAMP)targeting parasite microsatellites.Acta Tropica,141(B):368-371
Cao CM.2006.Tomato canker and its control.Inner Mongolia Agricultural Science and Technology,(5):70(in Chinese)[曹春梅.2006.番茄溃疡病的发生和防治.内蒙古农业科技,(5):70]
Du LL,Wang LJ,Zhou T,Lan Y,Fan YJ,Zhou YJ.2014.Comparison of detection methods for Southern rice black-streaked dwarf virus.Journal of Plant Protection,41(3):352-359(in Chinese)[杜琳琳,王跞姣,周彤,兰莹,范永坚,周益军.2014.南方水稻黑条矮缩病毒检测方法的比较.植物保护学报,41(3):352-359]
Duan YB,Xiao XM,Yang Y,Cao JH,Shi YY,Zhang XK,Wang Y,Wang JX,Zhou MG.2016.Establishment and application of a loop-mediated isothermal amplification method for detection of carbendazim-resistant mutants in Fusarium graminearum.Journal of Nanjing Agricultural University,39(1):97-105(in Chinese)[段亚冰,效雪梅,杨莹,曹君红,施一渊,张晓柯,王勇,王建新,周明国.2016.一种基于LAMP技术快速检测小麦赤霉病菌对多菌灵抗药性方法的建立及应用.南京农业大学学报,39(1):97-105]
Feng LP,Ni XM,Wei XT,Shao XL,Wu XH,Li Y.2013.Detection of Clavibacter michiganensis subsp.michiganensis by loop-mediated isothermal amplification.Journal of Food Safety and Quality,4(4):1207-1212(in Chinese)[封立平,尼秀媚,魏晓棠,邵秀玲,吴兴海,厉艳.2013.番茄溃疡病菌的环介导等温核酸扩增技术检测方法.食品安全质量检测学报,4(4):1207-1212]
Fu HY,Ge DF,Li XY,Wu XB,Chen RK,Gao SJ.2017.Nested-PCRdetection of Acidovorax avenae subsp.avenae,the pathogen of red stripe on sugarcane.Journal of Plant Protection,44(2):276-282(in Chinese)[傅华英,葛丹凤,李晓燕,吴小斌,陈如凯,高三基.2017.甘蔗赤条病菌巢式PCR检测.植物保护学报,44(2):276-282]
Luo LX,Zhao TC,Li JQ,Zhang L,Li Y,Hasan B.2004.Progress in research on bacterial canker of tomato caused by Clavibacter michiganensis subsp.michiganensis.Scientia Agricultura Sinica,37(8):1144-1150(in Chinese)[罗来鑫,赵廷昌,李健强,张乐,李勇,Hasan B.2004.番茄细菌性溃疡病研究进展.中国农业科学,37(8):1144-1150]
LüQY,Jiang N,Han SN,Li JQ,Luo LX.2017.Detection of Clavibacter michiganensis subsp.michiganensis in tomato seed by loop-mediated isothermal amplification based on virulence gene sequence.Acta Phytopathologica Sinica,47(5):630-639(in Chinese)[吕青阳,蒋娜,韩思宁,李健强,罗来鑫.2017.基于致病基因序列的LAMP方法检测番茄种子携带的溃疡病菌.植物病理学报,47(5):630-639]
Min XH,Liu J,Han YW,Wen ZH,Wang JP,Liu ST,Liu YL,Song R,Shi YB.2011.Nested-PCR for detection of seed-borne pathogen of tomato bacterial canker,Clavibacter michiganensis subsp.michiganensis.Acta Agriculturae Boreali-Occidentalis Sinica,20(7):28-31(in Chinese)[闵现华,刘箐,韩跃武,文朝慧,王军平,刘姝彤,刘雅莉,宋蕤,施颖波.2011.检测种传番茄细菌性溃疡病菌.西北农业学报,20(7):28-31]
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
Tong QB,Chen R,Zhang Y,Yang GJ,Kumagai T,Furushima-Shimogawara R,Lou D,Yang K,Wen LY,Lu SH,et al.2015.Anew surveillance and response tool:risk map of infected Oncomelania hupensis detected by loop-mediated isothermal amplification(LAMP)from pooled samples.Acta Tropica,141(B):170-177
Wang NW,Wang T,Shen JG,Hu FP.2014.Rapid detection for Clavibacter michiganensis subsp.michiganensis using real-time PCRbased on padlock probe.Scientia Agricultura Sinica,47(5):903-911(in Chinese)[王念武,王婷,沈建国,胡方平.2014.基于锁式探针的番茄溃疡病菌实时荧光PCR快速检测.中国农业科学,47(5):903-911]
Wei HY,Wang X,Li HM,Sun WR,Gu JF.2016.Loop-mediated isothermal amplification assay for rapid diagnosis of Meloidogyne mali.Journal of Plant Protection,43(2):260-266(in Chinese)[魏洪岩,王暄,李红梅,孙文荣,顾建锋.2016.采用环介导等温扩增法(LAMP)快速检测苹果根结线虫.植物保护学报,43(2):260-266]
Yasuharabell J,Kubota R,Jenkins DM,Alvarez AM.2013.Loop-mediated amplification of the Clavibacter michiganensis subsp.michiganensis micA gene is highly specific.Phytopathology,103(12):1220-1226
Zhao S,Li JY,Zhou Y,Yang WX,Gui YX,Zhang N,Liu DQ.2015.Development of a loop-mediated isothermal amplification assay for detection of Clavibacter michiganensis subsp.michiganensis.Journal of Agricultural University of Hebei,38(3):19-23(in Chinese)[赵赛,李建嫄,周颖,杨文香,贵亚欣,张娜,刘大群.2015.番茄溃疡病菌LAMP快速检测方法的建立.河北农业大学学报,38(3):19-23]
Zhao YQ,Tian YL,Luo JY,Yao HM,Yu H,Chen L,Hu BS.2015.Rapid diagnostic methods for Acidovorax citrulli using LAMP.Plant Protection,41(5):116-120(in Chinese)[赵玉强,田艳丽,罗金燕,姚红梅,余慧,陈磊,胡白石.2015.基于环介导等温扩增技术检测瓜类细菌性果斑病菌.植物保护,41(5):116-120]
Cao CM.2006.Tomato canker and its control.Inner Mongolia Agricultural Science and Technology,(5):70(in Chinese)[曹春梅.2006.番茄溃疡病的发生和防治.内蒙古农业科技,(5):70]
Du LL,Wang LJ,Zhou T,Lan Y,Fan YJ,Zhou YJ.2014.Comparison of detection methods for Southern rice black-streaked dwarf virus.Journal of Plant Protection,41(3):352-359(in Chinese)[杜琳琳,王跞姣,周彤,兰莹,范永坚,周益军.2014.南方水稻黑条矮缩病毒检测方法的比较.植物保护学报,41(3):352-359]
Duan YB,Xiao XM,Yang Y,Cao JH,Shi YY,Zhang XK,Wang Y,Wang JX,Zhou MG.2016.Establishment and application of a loop-mediated isothermal amplification method for detection of carbendazim-resistant mutants in Fusarium graminearum.Journal of Nanjing Agricultural University,39(1):97-105(in Chinese)[段亚冰,效雪梅,杨莹,曹君红,施一渊,张晓柯,王勇,王建新,周明国.2016.一种基于LAMP技术快速检测小麦赤霉病菌对多菌灵抗药性方法的建立及应用.南京农业大学学报,39(1):97-105]
Feng LP,Ni XM,Wei XT,Shao XL,Wu XH,Li Y.2013.Detection of Clavibacter michiganensis subsp.michiganensis by loop-mediated isothermal amplification.Journal of Food Safety and Quality,4(4):1207-1212(in Chinese)[封立平,尼秀媚,魏晓棠,邵秀玲,吴兴海,厉艳.2013.番茄溃疡病菌的环介导等温核酸扩增技术检测方法.食品安全质量检测学报,4(4):1207-1212]
Fu HY,Ge DF,Li XY,Wu XB,Chen RK,Gao SJ.2017.Nested-PCRdetection of Acidovorax avenae subsp.avenae,the pathogen of red stripe on sugarcane.Journal of Plant Protection,44(2):276-282(in Chinese)[傅华英,葛丹凤,李晓燕,吴小斌,陈如凯,高三基.2017.甘蔗赤条病菌巢式PCR检测.植物保护学报,44(2):276-282]
Luo LX,Zhao TC,Li JQ,Zhang L,Li Y,Hasan B.2004.Progress in research on bacterial canker of tomato caused by Clavibacter michiganensis subsp.michiganensis.Scientia Agricultura Sinica,37(8):1144-1150(in Chinese)[罗来鑫,赵廷昌,李健强,张乐,李勇,Hasan B.2004.番茄细菌性溃疡病研究进展.中国农业科学,37(8):1144-1150]
LüQY,Jiang N,Han SN,Li JQ,Luo LX.2017.Detection of Clavibacter michiganensis subsp.michiganensis in tomato seed by loop-mediated isothermal amplification based on virulence gene sequence.Acta Phytopathologica Sinica,47(5):630-639(in Chinese)[吕青阳,蒋娜,韩思宁,李健强,罗来鑫.2017.基于致病基因序列的LAMP方法检测番茄种子携带的溃疡病菌.植物病理学报,47(5):630-639]
Min XH,Liu J,Han YW,Wen ZH,Wang JP,Liu ST,Liu YL,Song R,Shi YB.2011.Nested-PCR for detection of seed-borne pathogen of tomato bacterial canker,Clavibacter michiganensis subsp.michiganensis.Acta Agriculturae Boreali-Occidentalis Sinica,20(7):28-31(in Chinese)[闵现华,刘箐,韩跃武,文朝慧,王军平,刘姝彤,刘雅莉,宋蕤,施颖波.2011.检测种传番茄细菌性溃疡病菌.西北农业学报,20(7):28-31]
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
Tong QB,Chen R,Zhang Y,Yang GJ,Kumagai T,Furushima-Shimogawara R,Lou D,Yang K,Wen LY,Lu SH,et al.2015.Anew surveillance and response tool:risk map of infected Oncomelania hupensis detected by loop-mediated isothermal amplification(LAMP)from pooled samples.Acta Tropica,141(B):170-177
Wang NW,Wang T,Shen JG,Hu FP.2014.Rapid detection for Clavibacter michiganensis subsp.michiganensis using real-time PCRbased on padlock probe.Scientia Agricultura Sinica,47(5):903-911(in Chinese)[王念武,王婷,沈建国,胡方平.2014.基于锁式探针的番茄溃疡病菌实时荧光PCR快速检测.中国农业科学,47(5):903-911]
Wei HY,Wang X,Li HM,Sun WR,Gu JF.2016.Loop-mediated isothermal amplification assay for rapid diagnosis of Meloidogyne mali.Journal of Plant Protection,43(2):260-266(in Chinese)[魏洪岩,王暄,李红梅,孙文荣,顾建锋.2016.采用环介导等温扩增法(LAMP)快速检测苹果根结线虫.植物保护学报,43(2):260-266]
Yasuharabell J,Kubota R,Jenkins DM,Alvarez AM.2013.Loop-mediated amplification of the Clavibacter michiganensis subsp.michiganensis micA gene is highly specific.Phytopathology,103(12):1220-1226
Zhao S,Li JY,Zhou Y,Yang WX,Gui YX,Zhang N,Liu DQ.2015.Development of a loop-mediated isothermal amplification assay for detection of Clavibacter michiganensis subsp.michiganensis.Journal of Agricultural University of Hebei,38(3):19-23(in Chinese)[赵赛,李建嫄,周颖,杨文香,贵亚欣,张娜,刘大群.2015.番茄溃疡病菌LAMP快速检测方法的建立.河北农业大学学报,38(3):19-23]
Zhao YQ,Tian YL,Luo JY,Yao HM,Yu H,Chen L,Hu BS.2015.Rapid diagnostic methods for Acidovorax citrulli using LAMP.Plant Protection,41(5):116-120(in Chinese)[赵玉强,田艳丽,罗金燕,姚红梅,余慧,陈磊,胡白石.2015.基于环介导等温扩增技术检测瓜类细菌性果斑病菌.植物保护,41(5):116-120]