转基因油菜TOPAS 19/2质粒分子多家协同实验及不确定度评定     

柳方方  张玲  宋贵文  曹应龙  盛灵慧  黄峥  王晶  秦培勇  卢长明 《中国农业科技导报》2012,1(1):31-35

质粒分子是转基因产品核酸定量检测的一类新型标准物质,具有易制备、周期短、成本低等特点。采用实时荧光定量PCR技术,并协同7家实验室对转基因油菜TOPAS 19/2质粒分子进行了基因组的可替代性研究、协同实验研究及不确定度评定。T检验表明,质粒和基因组所产生的内源和外源基因标准曲线的斜率和线性相关系数没有显著性差异。对多家定值的数据进行了统计分析得出,TOPAS19/2质粒分子的量值结果0.910,扩展标准不确定度（K=2）为0.013。  相似文献   

小型猪复合麻醉剂及其特异性颉颃剂交互作用对大鼠不同脑区iNOSmRNA转录的影响     

牛栋梁  王会英  姜胜  张士霞  范宏刚  王洪斌 《兽医大学学报》2014,(9):1491-1495

为研究小型猪专用复合麻醉剂（XFM）及其特异性颉颃剂交互应用对大鼠不同脑区iNOS mRNA转录的影响,将48只SD大鼠随机分为生理盐水对照组（C）和XFM与颉颃剂交互作用组（MJ）,MJ组分为早期交互（MJ1、MJ2）和晚期交互（MJ3、MJ4）2个亚组。各组大鼠到达预定时间点后分别采取脑组织,应用实时荧光定量PCR技术检测各组织中iNOS mRNA表达量。结果显示,XFM及其特异性颉颃剂交互应用时,大脑、小脑、海马、脑干和丘脑中iNOS mRNA转录均受到显著抑制（P〈0.01）,虽能逐渐回升,但在试验选取时间点内只有小脑与海马中iNOS mRNA转录恢复正常。结果表明,XFM及其特异性颉颃剂在交互作用时能够显著抑制中枢神经系统中iNOS mRNA的转录,部分脑区可以完全恢复,这可能与XFM及其特异性颉颃剂交互作用机制有关。  相似文献   

Distribution and properties of geographically distinct isolates of sugar beet yellowing viruses   总被引：1，自引：0，他引：1  

M. Stevens  N. J. Patron ‡  C. A. Dolby  R. Weekes §  P. B. Hallsworth  O. Lemaire  H. G. Smith 《Plant pathology》2005,54(2):100-107

From a total of 261 yellow sugarbeet leaves collected from 10 countries representing three continents, the incidence and distribution of strains of Beet mild yellowing virus (BMYV), Beet chlorosis virus (BChV) and Beet yellows virus (BYV) were analysed using serological and molecular methods. BMYV was found in all countries except Greece, and more frequently in the northern and western areas of Europe, whereas BYV predominated in Turkey, Spain, Greece, the USA and Chile. BChV, originally found in the USA and the UK in 1989, was identified in France, Spain, the Netherlands and Chile. Nine sugar beet poleroviruses, plus a reference isolate of Turnip yellows virus (TuYV, syn. Beet western yellows virus ), were further characterized and compared. Isolates obtained from sugar beet infected this species, but not oilseed rape or lettuce; all isolates except one infected Capsella bursa-pastoris . The coat-protein sequences of these isolates were highly similar, with the consensus sequence representing 89% of nucleotide residues. Within the coat-protein gene, two regions were identified that could represent specific epitopes to which monoclonal antibody BYDV-PAV-IL-1 could bind; this antibody is used to distinguish beet poleroviruses in ELISA. Comparison of the sequences at the 5' end showed that sequence homology existed only between isolates with the same host range. The first sequence data of polerovirus isolates from Chile are presented, showing that the coat protein and the 5' end of their genomes are highly similar to those of BMYV isolates found in Europe. Chilean polerovirus isolates may have been imported from the northern hemisphere in sugar beet breeding material.  相似文献   

苹果茎沟病毒部分分离物的生物学特性与分子鉴定          下载免费PDF全文

郑银英  王国平  洪霓 《植物保护学报》2005,32(3):266-270

通过昆诺藜接种鉴定和ELISA检测,从梨和苹果上分离获得苹果茎沟病毒(Apple stem grooving virus,ASGV)23个分离物.采用TC-RT-PCR对这些分离物进行扩增,均获得特异的扩增片段,PCR产物经5%PAGE电泳,出现大小约500、530和600bp的3种迁移率不同的泳动带型.根据PCR产物电泳迁移率的差异,选取3个来源于梨的分离物P-L4、P-6-1-17和P-3-2-67的PCR产物进行克隆与序列测定.经BLAST搜索,3个分离物的扩增片段与苹果分离物P-209的CP基因3′端核苷酸序列同源性分别为92.2%、90.4%和88.4%.3个分离物间的核苷酸序列也有较大差异,P-L4/P-6-1-17为95.5%、P-L4/P-3-2-67为90.4%、P-6-1-17/P-3-2-67为88.6%.  相似文献   

大豆制品转基因成分检测和定量标识分析     

董流昕  谢响明  毛建军  陈红运  陈洪俊  朱水芳 《植物检疫》2005,19(3):132-134

分别以大豆凝集素(Lectin)和5-莽草酸-3磷酸合成酶(CP4-EPSPS)为内源和目标基因,对28种大豆制品进行实时荧光PCR检测.检测结果表明,4种大豆制品的CP4-EPSPS扩增结果为阳性,含有转基因大豆成分.转基因成分的定量检测在贸易和可操作性方面要优于定性检测.  相似文献   

桃潜隐花叶类病毒RT-PCR和分子杂交检测技术的建立     

徐文兴  王国平洪霓 《植物检疫》2005,19(5):268-271

本研究从武汉周边采集表现典型花叶症状的桃样品,提取总RNA为模板,采用RT-PCR方法对这些样品进了分析,结果显示所分析的5个样品(P1～P5)均获得了预期大小约为337bp的目标扩增条带,表明样品均带有PLMVd.通过回收RT-PCR产物,用生物素标记制备探针,分别采用DNA斑点杂交、RNA斑点杂交和组织印迹杂交3种杂交方法对这些样品进行检测比较,3种杂交方法中,组织印迹杂交操作步骤相对简单快捷,适合于对PLMVd进行大田快速检测.  相似文献   

RT-PCR结合限制性内切酶分析法区分NDV强弱毒株   总被引：4，自引：0，他引：4  

耿捷  龚振华  李会荣  尹燕博  黄运生  罗卫  张彦明 《中国兽医学报》2005,25(5):463-466

通过设计的特异引物，采用RT-PCR对1996年以来分离的45株国内新城疫病毒分离株进行鉴定和分析，并与其MDT实验结果进行比较，建立了RT-PCR结合BglⅠ内切酶分析区分新城疫强弱毒的方法。此方法操作简便、迅速，不仅能区分新城疫的强弱毒株，还可诊断AMPV-1引起的其他禽类的感染。  相似文献   

Real-time PCR和常规PCR方法快速鉴定肉骨粉中的牛源性成份   总被引：3，自引：0，他引：3  

李林  陈继明  王志亮 《中国预防兽医学报》2005,27(1):50-54

疯牛病是一种严重危害动物和人类健康的疾病,其病原朊病毒可以通过食物链进行传播,因此建立准确的检测牛源性成份的方法非常重要.我们采用常规PCR和Real-time PCR中的TaqMan技术建立了快速鉴定牛源性成份的方法,使用Chelex-100提取肉骨粉中的DNA,过程简单可靠仅需要20min左右.对不同含量的牛肉骨粉进行检测,常规PCR能够检测到的最低含量是0.001%,而Real-time PCR对相同的样品进行检测所得到的灵敏度更高,最高可达到0.0001%,两种PCR方法的鉴定过程都非常简单快速,各需要2 h左右.这两种方法可以根据各实验室的不同条件作为肉骨粉鉴别的常规检测方法.  相似文献   

Identification of divergent variants of Grapevine virus A     

D.E. Goszczynski  A.E.C. Jooste 《European journal of plant pathology / European Foundation for Plant Pathology》2003,109(4):397-403

Eight isolates of Grapevine virus A (GVA), which induced different symptoms in leaves of Nicotiana benthamiana, were recovered from various grapevines. The dsRNA patterns of two isolates, which consistently induced mild vein clearing (referred here as mild isolates of GVA) were similar, but different from those of other isolates of GVA. Analysis based on overall nucleotide (nt) sequence identity in the 3 terminal part of the GVA genome, comprising part of ORF3 (putative movement protein, MP), entire ORF4 (capsid protein, CP), entire ORF5 and part of 3 UTR, revealed that GVA isolates separate into three groups (I, II, III), sharing 91.0–99.8% nt sequence identity within groups and 78.0–89.3% nt sequence identity between groups. Mild isolates of the virus were group III and shared only 78.0–79.6% nt sequence identity with the other isolates. The comparison of predicted amino acid sequences for MP and CP revealed many amino acid alterations, revealing distinct local net charges of these proteins for mild isolates of the virus. Based on both conserved and divergent nt regions in the CP and ORF5, oligonucleotide primers were designed for the simultaneous RT-PCR detection of all GVA isolates and for the specific detection of the most divergent virus variants represented here by mild isolates of the virus.  相似文献   

腹水综合征肉鸡肺脏缺氧诱导因子-1α基因克隆及表达研究   总被引：2，自引：0，他引：2  

曾秋凤  陈代文  张克英  余冰 《畜牧兽医学报》2006,37(6):609-613

从21日龄患腹水综合征（AS）鸡肺脏中提取RNA,根据健康鸡胚心室肌细胞缺氧诱导因子-1α（HIF-1α）基因序列（登录号为NM204297）设计引物,采用RT-PCR技术进行cDNA扩增,扩增产物经琼脂糖凝胶电泳分离、纯化、回收后与载体pMD18-T连接,转化大肠杆菌DH5α,筛选阳性克隆,测序.结果表明,已成功克隆的阳性重组子经测序鉴定其片段大小与预期大小完全一致,为1 576 bp.序列同源性分析比较表明,该cDNA及其推导的氨基酸序列与鸡胚HIF-1α cDNA和氨基酸序列同源性分别为99%和98%.采用RT-PCR技术检测4只21日龄正常肉鸡和4只21日龄AS患鸡肺脏HIF-1α mRNA,结果表明,AS患鸡肺脏HIF-1α mRNA表达丰度极显著高于正常肉鸡（P《0.01）.本试验证明,HIF-1α与肉鸡肺动脉高压导致的腹水综合征相关.  相似文献