番茄溃疡病菌实时荧光PCR快速检测方法研究   总被引：2，自引：0，他引：2  

吴兴海  邵秀玲  邓明俊  陈长法  厉艳  梁成珠 《江西农业学报》2007,19(3):34-36

根据番茄溃疡病菌ITS(16S~23SrDNA间隔区)基因序列,设计并合成了荧光PCR检测引物(Fan1和Fan2)及特异性检测探针(Fanprobe),对番茄溃疡病菌和非番茄溃疡病菌的标准菌株实时荧光PCR反应。结果表明,番茄溃疡病菌PCR产物出现强烈的特异杂交信号,而非番茄溃疡病菌均未出现特异荧光信号,证明这对引物及探针具有番茄溃疡病菌鉴定特异性。将番茄溃疡病菌DNA做梯度稀释,测定该检测体系的敏感度,结果表明,此体系可检出52.3 fg/μL的模板。  相似文献   

Microscopic analysis of plant–bacterium interactions using auto fluorescent proteins     

Guido V. Bloemberg 《European journal of plant pathology / European Foundation for Plant Pathology》2007,119(3):301-309

Plant growth promoting rhizobacteria (PGPR) include bacteria that fix nitrogen (e.g., Rhizobiaceae, Herbaspirillum, Azoarcus), produce phytohormones (e.g., Azospirillum) and provide protection against fungal and/or bacterial pathogens (e.g., Pseudomonas, Bacillus, Streptomyces). Interactions between PGPR and plants can be divided into different steps which include initial attraction, attachment, proliferation and colonization e.g., of roots, stem, leaves and flowers. At the genetic level the expression of many bacterial genes are altered during these processes. In addition to the interaction with the plant, PGPR interact and compete with the endogenous microflora, consisting of other bacteria, fungi and/or mycorrhizal fungi. In the case of biocontrol bacterial strains, a direct interaction with the pathogen is often required to suppress the disease. Microscopic analyses of plant growth promoting rhizobacteria (PGPR) in their natural environment and in specific during their interaction(s) with the host plant(s) and/or their target organism(s) is essential for the elucidation of their functioning and the successful application of commercial inoculants. With the discovery and development of auto fluorescent proteins (AFPs) as markers and the development of highly sophisticated fluorescence microscopes such as confocal laser scanning microscopes, a new dimension has been created for studying PGPR in their natural environment. This paper will give a short overview on available tools, the application of AFPs in PGPR research and some future perspectives. Several recent reviews will give the reader an option for further reading (Bloemberg and Lugtenberg 2004; Chalfie and Kain 2005; Larrainzar et al. 2005; Rediers et al. 2005; Bloemberg and Camacho 2006).  相似文献   

实时荧光PCR技术快速定量检测H5亚型禽流感病毒     

韩剑锋  宁宜宝  宋立  杨承槐 《中国预防兽医学报》2007,29(6):453-458

选取H5亚型禽流感病毒血凝素(Hemagglutinin,HA)基因保守序列,使用Primer Express 2.0软件设计出特异性引物和TaqMan MGB探针,利用实时荧光PCR技术来定量检测禽流感病毒。使用含有选定检测序列的RNA标准品做标准曲线,结果表明该方法的灵敏度为10拷贝/反应,标准曲线的相关系数为0.998003,对H9亚型禽流感病毒和其他禽病病毒无交叉反应,特异性好、重复性佳。为禽流感病毒检测提供了一种特异、敏感、快速的定量检测方法。  相似文献   

应用TaqMan MGB探针检测小麦印度腥黑穗病菌和黑麦草腥黑穗病菌   总被引：1，自引：0，他引：1  

章桂明  程颖慧  王颖  杨伟东  易建平 《植物病理学报》2006,36(2):142-151

 以小麦印度腥黑穗病菌9个菌株和黑麦草腥黑穗病菌5个菌株及其近似种或相关种:稻粒黑粉菌、狼尾草腥黑粉菌、狗尾草腥黑粉菌、苏玛特腥黑粉菌、狐尾草腥黑粉菌、小麦网腥黑穗病菌和小麦矮化腥黑穗病菌共9种22个菌株为研究对象,通过序列比对分析,设计了检测小麦印度腥黑穗病菌及黑麦草腥黑穗病菌的TaqMan MGB实时荧光PCR引物和探针,优化了反应条件,筛选出特异性探针,分别建立了小麦印度腥黑穗病菌和黑麦草腥黑穗病菌实时荧光单重PCR和实时荧光双重PCR检测方法,其中实时荧光双重PCR检测方法实现了在同一PCR管中仅用5μL的反应体系,进行1次PCR反应就能特异性检测出小麦印度腥黑穗病菌或黑麦草腥黑穗病菌。本研究所建立的检测方法特异性强、结果可靠、检测速度快、成本明显降低,在文际应用中具有推广价值。  相似文献   

实时荧光RT-PCR一步法检测番茄环斑病毒   总被引：16，自引：1，他引：16  

朱建裕  朱水芳  廖晓兰  高必达 《植物病理学报》2003,33(4):338-341

 根据番茄环斑病毒(ToRSV)各株系RNA1聚合酶基因的保守序列,设计并合成1对引物和1条TaqMan荧光探针,建立了对ToRSV的实时荧光RT-PCR检测方法。该方法利用TaqMan荧光探针实时监测目的基因的扩增,实现RT-PCR扩增与探针检测同时进行,不需PCR后处理,消除了PCR产物的污染,不需电泳和EB染色,减少了检测步骤,节省了时间。这个方法具有"灵敏、准确、简便、快速"的特点适合于种传病害的快速诊断和口岸检验检疫运用。  相似文献   

苜蓿萎蔫病菌TaqMan探针实时荧光PCR检测方法的建立   总被引：15，自引：1，他引：15  

漆艳香  赵文军  朱水芳  肖启明  廖晓兰  陈红运 《植物检疫》2003,17(5):260-264

苜蓿萎蔫病菌是我国对外检疫性二类有害生物，目前国内尚无发生6在出入境捡验检疫中主要是采用生物学和血清学方法进行检测，劳动强度大，耗费时间长。根据苜蓿萎蔫病菌与其它细菌菌株16SrDNA序列差异，设计出对苜蓿萎蔫病菌具有稳定点突交特异性探针，利用该探针对棒形杆菌属4个种及其它属细菌进行了实时荧光PCR检测实验。结果表明，只有苜蓿萎蔫病菌能检测到荧光信号，其它细菌没有荧光产生。该方法特异性强，灵敏度高，能检测到21．4fg质粒DNA，比常规PCR灵敏100倍，而且整个过程只需要2～3h。该方法可有效地应用于进出境病原菌检测之中。  相似文献   

Construction of recombinant adenovirus expressing BDNF and its expression in expanded rat mesenchymal stem cells in vitro     

LI Hong-le  LI Hao-wei  XING Fei-yue  SUN Xue-gang  DENG Yu-bin  ZHANG Xiu-ming  JANG Yong  LI Shu-nong 《园艺学报》2003,19(4):438-442

AIM:To construct recombinant adenovirus vector containing brain derived neurotrophic factor, (BDNF) gene using bacterial homogenous recombination, and investigate the expression in expanded rat mesenchymal stem cells (rMSC) in vitro.METHODS:BDNF gene and proBDNF gene were subcloned into adenovirus shuttle plasmid pAdTrack-CMV containing enhanced green fluorescent protein gene (EGFP) expression cassette, forming shuttle vector of pAdTrack-BDNF, and pAdTrack-proBDNF, and co-transformed into BJ5183 bacterial cells with adenovirus backbone vector pAdEasy-1 using chemical transformation. After the recombinant adenovirus vector was obtained, the identified recombinant adenovirus plasmid DNA was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. rMSC were infected by recombinant adenovirus and EGFP expression was detected using fluorescent microscope. Infection efficiency was assessed by flow cytometrics. Western blotting identified expression of Ad -proBDNF and Ad-BDNF in rMSC. rMSC infected with Ad -proBDNF and Ad-BDNF were induced to differentiate into neuron-like cells. rMSC infected with Ad -proBDNF and Ad-BDNF were injected into nude mice and assessd in vivo.RESULTS:We successfully constructed the recombinant adenovirus Ad -proBDNF and Ad-BDNF that expressed in expanded rMSC in vitro.CONCLUSION:Recombinant adenovirus high-effectively mediates Ad -proBDNF and Ad-BDNF expression in expanded rMSC in vitro and in vivo.  相似文献   

The Use of Molecular Beacons Combined with NASBA for the Sensitive Detection of Sugarcane Yellow Leaf Virus     

M.C. Goncçalves  M.M. Klerks  M. Verbeek  J. Vega  J.F.J.M. van den Heuvel 《European journal of plant pathology / European Foundation for Plant Pathology》2002,108(5):401-407

Sugarcane yellow leaf virus (ScYLV) is widely distributed in Brazil and other sugarcane producing countries causing significant yield losses. Due to the high incidence of the aphid vector, the virus is widespread in the field and in parental clones used in sugarcane breeding programmes. Aiming to present a sensitive and reliable detection of ScYLV, we have adapted an AmpliDet RNA system, compared it with the currently available detection methods and discussed its applicability for routine diagnosis. AmpliDet RNA consists of nucleic acid sequence-based amplification (NASBA) of the target RNA with specific primers and simultaneous real-time detection of the amplification products with molecular beacons. The results showed that the system produced a detection level of at least 100fg of purified virus. Virus was readily detected in plant tissues with low levels of infection (without the need of previous RNA extraction) and in the hemolymph of aphids. The method showed to be virus-specific, testing negative for other species of the Luteoviridae. In conclusion, the system has potential to become a diagnostic method for the detection of sugarcane viruses.  相似文献   

Induction of systemic resistance to<Emphasis Type="Italic">Colletotrichum lindemuthianum</Emphasis> in bean by a benzothiadiazole derivative and rhizobacteria   总被引：1，自引：0，他引：1  

J. Bigirimana  M. Höfte 《Phytoparasitica》2002,30(2):159-168

Plants have developed mechanisms to resist secondary infection upon inoculation with a necrotizing pathogen, chemical treatment as well as treatment with some non-pathogenic microorganisms such as rhizosphere bacteria. This phenomenon has been variously described as induced systemic resistance (ISR) or systemic acquired resistance. In the present study, the chemical benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester (BTH, acibenzolar-S-methyl), and the rhizobacteriaPseudomonas aeruginosa KMPCH andP. fluorescens WCS417 were tested for their ability to induce resistance toColletotrichum lindemuthianum in susceptible and moderately resistant bean plants (Phaseolus vulgaris L.). BTH induced local and systemic resistance when bean leaves were immersed in 10⁻³ to 10⁻⁷ M BTH 3 days before the challenge inoculation. At a high concentration (10⁻³ M), BTH induced resistance of the same order as resistance induced by the pathogenC. lindemuthianum, although at this high concentration BTH appeared to be phytotoxic. Soil and seed treatment with 1 mg kg⁻¹ BTH protected beans against anthracnose. BTH-mediated induced resistance was effective in susceptible and moderately resistant plants.P. aeruginosa KMPCH induced resistance in bean againstC. lindemuthianum only in a moderately resistant interaction. KMPCH-567, a salicylic acid mutant of KMPCH, failed to induce resistance, indicating that salicylic acid is important for KMPCH to induce resistance in the bean—C. lindemuthianum system.P.fluorescens WCS417 could induce resistance toC. lindemuthianum in a susceptible and in moderately resistant interactions. http://www.phytoparasitica.org posting Jan. 16, 2002.  相似文献   

曼氏无针乌贼荧光标记技术初步研究     

董智勇  吴常文  叶德锋 《浙江水产学院学报》2010,(2):120-128

通过在曼氏无针乌贼幼体（胴长2.0-3.0cm）背部注射浓度为350mg/L的茜素络合指示剂（Alizarin Complex-one,ALC）溶液,以实现对其内壳的染色标记。将标记组与对照组幼体各125头饲养于同一养殖试验池（370cm×370cm×140cm）内,并分别于标记后的第0d、10d、20d、30d、40d、50d对2组个体进行随机取样,测量其胴长、胴宽、体重、壳长、壳宽、壳重6个生物学指标,观察内壳标记色保持情况并记录死亡率。将结果进行方差分析后显示,荧光注射标记方法对曼氏无针乌贼的成活率和生长发育均没有显著影响（P〉0.05）;至乌贼发育成熟并产卵时,紫红色的椭圆形标记依然清晰可见,从外部透过皮肤也具有可视的直观效果,保持率为100%。荧光注射标记法适用于曼氏无针乌贼的标记,具有成本低、操作简单、标记色易识别等优点,可进行批量标记试验。  相似文献