Optimized quantification of unculturable Candidatus Liberibacter spp. in host plants using real-time PCR
Optimized quantification of unculturable Candidatus Liberibacter spp. in host plants using real-time PCR
复制标题
DOI：
10.1094/pdis-92-6-0854
复制
发表时间：
2008-06-01
期刊：
影响因子：
4.5
通讯作者：
Levy, Laurene
中科院分区：
文献类型：
--
作者：
Li, Wenbin;Li, Dayan;Levy, Laurene
柑桔黄龙病（Citrus huanglongbing，HLB）是由韧皮部限制性木虱介导的柑桔黄龙病菌（Citrus Liberibacter spp.）并且是重要性迅速增加的柑橘的破坏性疾病。2004年在巴西的圣保罗和2005年在佛罗里达的主要柑橘产区报道了该病。已经开发了各种实验室方法来确认基于病原体的疾病诊断或检测或鉴定病原体;然而，由于病原体在人工培养基中仍然不可培养，因此在宿主或载体相互作用中没有关于病原体滴度的定量信息。我们以前开发了一种定量聚合酶链反应（PCR）为基础的检测钙。韧皮杆菌属在这项研究中，我们评估了样品组成对TaqMan实时PCR定量柑橘植物中病原体的影响。使用含有来自病原体的靶DNA的克隆质粒和来自田间生长的HL B感染的柑橘植物的总DNA样品建立标准曲线。回归分析表明，从自然感染的田间生长的植物中提取的DNA建立的标准曲线比从含有扩增靶作为克隆插入物的质粒构建的标准曲线更准确。非靶DNA和假定的PCR抑制剂从柑橘植物的实时PCR的灵敏度和扩增效率下降时，质粒提供的模板靶在“加标”健康柑橘DNA提取物。这种效应在植物组织类型、柑橘物种和地理位置之间存在差异。基于这些样品效应，已经建立了用于在不同地理位置种植的不同柑橘物种的各种柑橘组织中定量病原体的通用标准曲线。PCR检测前样品在4 ℃下储存2个月不影响随后的病原体定量。经验证的定量实时PCR方法和通用标准曲线将是非常有用的宿主-病原体相互作用和流行病学的研究，并在疾病的控制策略的发展。
Citrus huanglongbing (HLB) is caused by the phloem-limited and psyllid-vectored Candidatus Liberibacter spp. and is a destructive disease of citrus that is rapidly increasing in importance. The disease was reported recently in the principle citrus-producing areas of Sao Paulo, Brazil in 2004 and in Florida in 2005. A variety of laboratory methods have been developed to confirm a symptom-based disease diagnosis or for the detection or identification of the pathogen; however, no quantitative information has been available on the pathogen titer in either host or vector interactions because the pathogen remains unculturable in artificial media. We previously developed a quantitative polymerase chain reaction (PCR)-based assay for detection of Ca. Liberibacter spp. and, in this study, we evaluated the effects of sample composition on quantification of the pathogen in citrus plants by TaqMan real-time PCR. Standard curves were established using cloned plasmids containing target DNA from the pathogen and with total DNA samples from field-grown HLB-infected citrus plants. Regression analysis showed that a standard curve established with DNA extracted from naturally infected field-grown plants was more accurate than the standard curve constructed from plasmids containing the amplification targets as cloned inserts. Nontarget DNA and putative PCR inhibitors from citrus plants decreased the sensitivity and the amplification efficiency of real-time PCR when plasmids provided the template target in "spiked" healthy citrus DNA extracts. This effect varied among plant tissue types, citrus species, and geographic locations. Based on these sample effects, a universal standard curve has been established for quantification of the pathogen in various citrus tissues of different citrus species planted in different geographic locations. Sample storage at 4 degrees C for 2 months prior to PCR assay did not affect subsequent quantification of the pathogen. The validated quantitative real-time PCR method and the universal standard curve will be very useful for studies of host-pathogen interactions and epidemiology, and in the development of control strategies for the disease.