柑橘黄龙病、裂皮病和衰退病病原的多重RT-PCR检测  

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

作　　者：丁芳[1] 曹庆[2] 王国平[1] 易干军[2] 钟云[2] 

机构地区：[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

出　　处：《园艺学报》2006年第5期947-952,共6页Acta Horticulturae Sinica

基　　金：国家863课题(2001AA241142);广东省科技攻关资助项目(2003B21601);湖北省攻关计划(2006AA203B05)

摘　　要：建立了一种同时检测柑橘黄龙病病原类细菌(CandidatusLiberibacterasiaticus,HLB)、柑橘裂皮类病毒(Citrusexocortisviroid,CEVd)、柑橘衰退病病毒(Citrustristezavirus,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RT-PCR扩增,得到1160bp、371bp、273bp3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RT-PCR技术体系。该体系最低能从10pg总RNA中检测出黄龙病类细菌和柑橘裂皮类病毒,从1pg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。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 this multiplex 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 multiplex RT-PCR system, 37 samples collected from field were tested. The results showed： two or three pathogens mix-infection was in existence in fields.

关 键 词：柑橘 多重RT-PCR 黄龙病 裂皮病 衰退病 检测 

分 类 号：S666[农业科学—果树学]