柑橘黄龙病常规PCR检测技术研究与初步应用
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摘要
柑橘黄龙病是世界范围具有毁灭性危害的柑橘细菌性病害,在我国大部分柑橘产区发生,严重制约了我国柑橘产业的发展。本文根据黄龙病病原物亚洲韧皮杆菌核糖体16SrRNA基因设计了1对PCR引物HLBF468/HLBR877,并以此为基础建立了常规PCR反应体系,确定了检测体系的特异性和灵敏度。结果表明该体系的检测灵敏度比先前报道的常规PCR方法有了明显提高。利用建立的PCR体系完成了对广东和广西两省区果园柑橘黄龙病的抽样检测。本研究建立的常规PCR方法可以作为一种简便、准确、灵敏的检测技术应用于柑橘黄龙病的早期诊断。
Citrus Huanglongbing(HLB)is a devastating bacterial disease on citrus in the world and seriously restricts the development of citrus industry in our country.Polymerase chain reaction(PCR)approach was established based on a pair of primers,HLBF468/HLBR877,designed from the ribosomal gene sequence of 16 S rRNA of Candidatus Liberibacter asiaticus,and its sensitivity was obviously higher than that of the previous reported PCR approach.The samples infected by HLB were collected from Guangdong and Guangxi and were tested by this method.The results showed that this method could be applied for early diagnosis of HLB.
Citrus Huanglongbing(HLB)is a devastating bacterial disease on citrus in the world and seriously restricts the development of citrus industry in our country.Polymerase chain reaction(PCR)approach was established based on a pair of primers,HLBF468/HLBR877,designed from the ribosomal gene sequence of 16 S rRNA of Candidatus Liberibacter asiaticus,and its sensitivity was obviously higher than that of the previous reported PCR approach.The samples infected by HLB were collected from Guangdong and Guangxi and were tested by this method.The results showed that this method could be applied for early diagnosis of HLB.
引文
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[4]许美容,陈燕玲,邓晓玲.柑橘黄龙病症状与“Candidatus Liberibacter asiaticus”PCR检测结果的相关性分析[J].植物病理学报,2016,46(3):367-373.
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[9]郭丽英,郭雁君,麦丽瑶,等.肇庆、云浮地区砂糖橘黄龙病PCR快速检测技术的建立[J].广东农业科学,2014(6):96-98.
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[11]胡龙娇,伍建榕,叶维雁,等.云南普文葡萄柚黄龙病病原分子检测及鉴定[J].中国南方果树,2015,44(2):14-17.
[12]王红,张秋明,王春梅.湘南地区柑橘黄龙病的PCR检测[J].湖南农业大学学报(自然科学版),2007,33(2):164-166.
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[16]孟祥春,钟云,刘岩,等.柑桔黄龙病PCR快速检测技术的建立与应用[J].西北植物学报,2007,27(7):1461-1463.
[17]张伟.柑橘黄龙病PCR检测技术研究[J].生物灾害科学,2012,35(2):164-168.
[18]王春梅,张秋明,王红.柑橘黄龙病PCR检测技术研究[J].湖南农业科学,2013(15):13-15.
[19]LI Wenbin,HARTUNG J S,LEVY L.Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus Huanglongbing[J].Journal of Microbiological Methods,2006,66(1):104-115.
[20]KAWAI T,KIKUKAWA K,SASAKI T,et al.Technique for detection of Huanglongbing using nested-PCR[J].Research Bulletin of the Plant Protection Service(Japan),2007(43):27-32.
[21]DENG Xiaoling,ZHOU Gen,LI Huaping,et al.Detection of Candidatus Liberibacter asiaticus from Wampee(Clausena lansium Skeels)by nested PCR[J].Plant Health Progress,2007,10:1094.
[22]邓崇岭,陈传武,赵小龙,等.用Nested-PCR检测广西黄皮与九里香黄龙病病原[J].中国农学通报,2010,26(17):273-276.
[23]肖远辉,陈国平,傅翠娜.柑橘黄龙病实时荧光PCR鉴定技术研究[J].安徽农业科学,2011,39(6):3339-3340.
[24]黄宏明,王茜,徐宁,等.广西砂糖橘黄龙病亚洲种的NestedPCR检测[J].南方农业学报,2013,44(12):1997-2000.
[2]李雪燕,项宇,胡文召.中国柑橘黄龙病发生情况及防治现状[J].中国植保导刊,2011,31(4):14-16.
[3]COLETTA-FILHO H D,CARLOS E F,ALVES K C S,et al.In planta multiplication and graft transmission of‘Candidatus Liberibacter asiaticus’revealed by real-time PCR[J].European Journal of Plant Pathology,2010,126(1):53-60.
[4]许美容,陈燕玲,邓晓玲.柑橘黄龙病症状与“Candidatus Liberibacter asiaticus”PCR检测结果的相关性分析[J].植物病理学报,2016,46(3):367-373.
[5]高玉霞,卢占军,钟八莲,等.柑桔黄龙病常规PCR检测4对引物的特异性和灵敏度[J].中国南方果树,2014,43(1):5-8.
[6]欧阳冬梅,蒋军喜,宋水林,等.应用PCR和测序技术诊断吉安和抚州两地区的柑橘黄龙病[J].江西农业大学学报,2009,31(6):1035-1038.
[7]张利平,陈国庆,王海琴,等.浙江柑橘黄龙病病原β-操纵子核糖体蛋白基因的PCR检测及其序列比对[J].植物保护,2010,36(1):80-82.
[8]王春梅,张秋明,王红.湖南岳阳地区柑橘黄龙病PCR检测[J].中国农学通报,2014,30(10):303-306.
[9]郭丽英,郭雁君,麦丽瑶,等.肇庆、云浮地区砂糖橘黄龙病PCR快速检测技术的建立[J].广东农业科学,2014(6):96-98.
[10]杨毅,车海彦,曹学仁,等.海南柑橘黄龙病PCR检测分析[J].甘肃农业,2015(24):50-51.
[11]胡龙娇,伍建榕,叶维雁,等.云南普文葡萄柚黄龙病病原分子检测及鉴定[J].中国南方果树,2015,44(2):14-17.
[12]王红,张秋明,王春梅.湘南地区柑橘黄龙病的PCR检测[J].湖南农业大学学报(自然科学版),2007,33(2):164-166.
[13]DO CARMO TEIXEIRA D,DANET J L,EVEILLARD S,et al.Citrus Huanglongbing in Sao Paulo State,Brazil:PCR detection of the‘Candidatus’Liberibacter species associated with the disease[J].Molecular and Cellular Probes,2005,19(3):173-179.
[14]TEIXEIRA D C,LOPES S A,YAMAMOTO P T,et al.PCR detection of the two Liberibacter species associated with citrus huanglongbing in S2o Paulo State,Brazil[C]//HILF M E,DURAN-VILA N,ROCHA-PEA M A.16th International Organization of Citrus Virologist Conference Proceedings.IOCV,Riverside,CA.2005:432-438.
[15]胡浩,殷幼平,张利平,等.柑桔黄龙病的常规PCR及荧光定量PCR检测[J].中国农业科学,2006,39(12):2491-2497.
[16]孟祥春,钟云,刘岩,等.柑桔黄龙病PCR快速检测技术的建立与应用[J].西北植物学报,2007,27(7):1461-1463.
[17]张伟.柑橘黄龙病PCR检测技术研究[J].生物灾害科学,2012,35(2):164-168.
[18]王春梅,张秋明,王红.柑橘黄龙病PCR检测技术研究[J].湖南农业科学,2013(15):13-15.
[19]LI Wenbin,HARTUNG J S,LEVY L.Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus Huanglongbing[J].Journal of Microbiological Methods,2006,66(1):104-115.
[20]KAWAI T,KIKUKAWA K,SASAKI T,et al.Technique for detection of Huanglongbing using nested-PCR[J].Research Bulletin of the Plant Protection Service(Japan),2007(43):27-32.
[21]DENG Xiaoling,ZHOU Gen,LI Huaping,et al.Detection of Candidatus Liberibacter asiaticus from Wampee(Clausena lansium Skeels)by nested PCR[J].Plant Health Progress,2007,10:1094.
[22]邓崇岭,陈传武,赵小龙,等.用Nested-PCR检测广西黄皮与九里香黄龙病病原[J].中国农学通报,2010,26(17):273-276.
[23]肖远辉,陈国平,傅翠娜.柑橘黄龙病实时荧光PCR鉴定技术研究[J].安徽农业科学,2011,39(6):3339-3340.
[24]黄宏明,王茜,徐宁,等.广西砂糖橘黄龙病亚洲种的NestedPCR检测[J].南方农业学报,2013,44(12):1997-2000.