作者：王中康,孙宪昀,夏玉先,周常勇,殷幼平
作者单位：重庆大学基因工程研究中心,西南农业大学植保学院大学基因工程研究中心,中国农业科学院柑桔研究所
摘要：柑桔溃疡病是严重影响全世界柑桔生产的重大检疫性病害,根据柑桔溃疡病菌(Xanthomonas axonopodis pv.citri)新近公布的全基因组中独有的保守蛋白基因序列,设计筛选出一对种特异性引物(JYF5/JYR5),能专一地扩增检出柑桔组织表面所带溃疡病菌的DNA靶带(413 bp)。而柑桔叶面附生的非致病性黄单胞菌、野油菜黄单胞菌近缘种以及健康柑桔样品都不能扩增;靶细菌DNA检测下限1.56 pg/μL,靶细菌悬浮液检测下限10 cfu/μL;在不同PCR仪及各种控温方式下都能稳定地扩增出特征性靶带。这一特异、准确的柑桔溃疡病菌PCR检验技术和研制的预包被固相化PCR检测试剂盒已开始用于我国非疫生产区建设中柑桔苗木、果实的病害检疫检验。
关键词：Xanthomonas axonopodis pv.citri,柑桔溃疡病,PCR,检疫
Title: Rapid detection of citrus bacterial canker disease (Xanthomonas axonopodis pv. citri) with polymerase chain reaction (PCR)
Authors: WANG Zhong-kang, SUN Xian-yun, XIA Yu-xian,ZHOU Chang-yong, YIN You-ping
Abstract: Citrus bacterial canker disease (Xanthomonas axonopodis pv. citri) is now still on the list of quarantine pest of international and Chinese administration. One pair of specific primers (JYF5/JYR5) designed from published sequence based on encoding a conserved hypothetical protein gene in genome of Xac (Nature, 2002) was screened and developed for specifically detecting different pathovars of Xac and citrus leaves and fruit with suspicious symptom of CBCD as well as healthy leaves of citrus spiked with pathogen of Xac. The specification of primer pair is better than the previous ones designed from plasmid DNA or from 16 S rDNA sequences of Xac. PCR amplification product of 413 bp target band was generated from target pathogen but not from non-pathogenic xanthomonads of citrus epiphytes and several plant pathogenic bacteria as well as from soaking solution of healthy citrus tissue. Limit of detection was 10 cells or 1. 56 pg per micro liter per reaction. Stabilized results were obtained using different type of thermocyclers with corresponding PCR profiles. The accurate and rapid molecular diagnosis technique was applied in the eradication program and for epidemiological purposes in maintenance and protection of non-quarantine area of citrus production was established. It is easy to use and will promote standardization of reaction process based on detection kit of PCR with pre-coated solidly reagents and simplified sample preparation procedure.
Key words: Xanthomonas axonopodis pv. citri； citrus bacterial canker disease； PCR； quarantine；
文献注录：王中康,孙宪昀,夏玉先,周常勇,殷幼平. 柑桔溃疡病菌PCR快速检验检疫技术研究 [J]. 植物病理学报. 2004, 01: 14-20.
基金: 农业部农业技术推广中心专项课题(2002-110);教育部“春晖计划”教外司留2003-589-18资助
报/刊名：《植物病理学报》，发表于2004 / 第 01 期。
文献类型： [J] （文献级别：核心刊物）
页码：第 14-20 页 / 共7 页