柑橘黄龙病、裂皮病和衰退病病原的多重RT-PCR检测

园艺学报 ›› 2006, Vol. 33 ›› Issue (5): 947-952.

柑橘黄龙病、裂皮病和衰退病病原的多重RT-PCR检测

丁芳^{1, 2};曹庆²;王国平^1*;易干军^2*;钟云²

(¹华中农业大学植物科学技术学院, 湖北武汉430070, ² 广东省农业科学院果树研究所, 广东广州510640)

收稿日期:2006-01-23 修回日期:2006-03-09 出版日期:2006-10-25 发布日期:2006-10-25

Studies on the Simultaneous Detection of Citrus Huanglongbing Pathogen,Citrus exocortis viroid, Citrus tristeza virus by Multiplex RT-PCR

Ding Fang^{1, 2};Cao Qing²;Wang Guoping^1*;Yi Ganjun^2*;Zhong Yun²

(¹Plant Science and Technology Academy, Huazhong Agricultural University, Wuhan, Hubei 430070, China;²Insititute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou, Guangdong 510640, China)

Received:2006-01-23 Revised:2006-03-09 Online:2006-10-25 Published:2006-10-25

摘要/Abstract

摘要： 建立了一种同时检测柑橘黄龙病病原类细菌(Candidatus L iberibacter asiaticus, HLB) 、柑橘裂皮类病毒(Citrus exocortis viroid, CEVd) 、柑橘衰退病病毒(Citrus tristeza virus, CTV) 的多重RT-PCR技术体系。运用根据3 种病原核苷酸保守区序列设计的特异性引物, 成功的对同一样品中的HLB、CEVd、CTV进行多重RT2PCR扩增, 得到1 160 bp、371 bp、273 bp 3条特异性大小与试验设计相符的条带。就影响多重PCR 的主要因素引物浓度和退火温度进行了一系列优化, 建立了能同时检测HLB、CEVd、CTV的多重RT2PCR技术体系。该体系最低能从10 pg总RNA中检测出黄龙病类细菌和柑橘裂皮类病毒, 从1 pg总RNA中检测到柑橘衰退病毒。对采自田间37份材料的实际检测表明: 存在两种或3种病原的复合侵染。

关键词: 柑橘, 多重RT-PCR, 黄龙病, 裂皮病, 衰退病, 检测

Abstract: A multiplex RT-PCR was successfully established to simultaneously detect Citrus Huanglongbing pathogen (Candidatus Liberibacter asiaticus, HLB) , Citrus exocortis viroid (CEVd) and Citrus tristeza virus (CTV). Using three sets of specific primers designed according to the converted sequences of these three
pathogens respectively, expected fragments of 1 160 bp (HLB) , 371 bp (CEVd) and 273 bp (CTV) were successfully amplified by thismultiplex RT-PCR systerm. To optimize the multiplex RT-PCR, the concentration of primers and annealing temperature were repeatedly tried. Final results showed that a multiplex RT-PCR system to simultaneously detect HLB, CEVd and CTV was firstly established successfully. Sensibility test showed: it could detect out HLB, CEVd in 10 pg, and CTV in 1 pg total RNA respectively. To confirm the utilization of the multip lex RT-PCR system, 37 samples collected from field were tested. The results showed:two or three pathogensmix2infection was in existence in fields.

Key words: Citrus, Multiplex RT-PCR, Huanglongbing, Citrus exocortis viroid, Citrus tristeza virus, Detection

丁芳;;曹庆;王国平;易干军;钟云. 柑橘黄龙病、裂皮病和衰退病病原的多重RT-PCR检测[J]. 园艺学报, 2006, 33(5): 947-952.

Ding Fang;;Cao Qing;Wang Guoping;Yi Ganjun;Zhong Yun. Studies on the Simultaneous Detection of Citrus Huanglongbing Pathogen,Citrus exocortis viroid, Citrus tristeza virus by Multiplex RT-PCR[J]. ACTA HORTICULTURAE SINICA, 2006, 33(5): 947-952.

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