A Universal `One-tube' RT-PCR Protocol for Amplifying Isolates of Bovine Viral Diarrhoea Virus     

Pfeffer M  Freyburg MV  Kaaden OR  Beer M 《Veterinary research communications》2000,24(7):491-503

The increase in the knowledge of the genetic variability of BVDV and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. This study was undertaken to develop a standard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction. The reaction set-up is very flexible because it consists of two pre-mixes. These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any primer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amounts sufficient for subsequent sequencing reactions. The method was applied to five different regions of the BVDV genome: (i) the well-known 5-UTR to differentiate genotypes I and II; (ii) the entire E2 gene, or an approximately 550 bp region within the E2 gene, in order to find the molecular equivalent of antigenic varieties; (iii) the entire structural protein coding region covering the Npro, capsid, E ^RNS, E1 and E2 genes; (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic biotypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B genes. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from either 10⁶ infected bovine embryonal lung cells or the same number of leukocytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis virus/BVDV recombinants. The correct processing of the amplicon-derived E2 glycoprotein of BVDV strain PT810 was demonstrated by its reaction with a monoclonal antibody in an immunofluorescence assay. Given the variety of RT-PCRs tested, we conclude that this universal protocol may be useful with other RNA viruses.  相似文献   

羊驼Cytb基因序列的测定及其研究   总被引：3，自引：0，他引：3  

董常生  高莉  贺俊平  赫晓燕  耿建军  赵英虎 《畜牧兽医学报》2006,37(12):1254-1258

测定了羊驼线粒体DNA细胞色素b基因部分序列，并与骆驼科其它物种细胞色素b基因进行同源序列比较，分析了碱基组成及变异情况，并用邻接法和最大简约法构建了分子系统树，在分子水平上探讨了羊驼在骆驼科动物中的分类地位。结果表明，羊驼与骆马和美洲驼存在的遗传差异相对比羊驼与原驼大，为传统分类学中的羊驼是原驼驯化以后的一种家养驼的提法提供了分子学依据。  相似文献   

Sequence analysis of the genome of OP2, a lytic bacteriophage of Xanthomonas oryzae pv. oryzae     

Yasuhiro Inoue  Takayuki Matsuura  Tatsuji Ohara  Koji Azegami 《Journal of General Plant Pathology》2006,72(2):104-110

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禽腺联病毒全基因组的克隆及感染性病毒的拯救     

王建业  孙怀昌  朱国强 《中国预防兽医学报》2007,29(11):825-829

为了克隆禽腺联病毒(Avian adeno-associated virus,AAAV)全基因组用于构建基因转移载体研究,以鸡胚致死孤儿病毒(CELO)作为辅助病毒与AAAV共接种SPF鸡胚进行AAAV的增殖,将AAAV约4.7kb双链基因组DNA与pCR2.1载体连接,构建了含AAAV全基因组的重组质粒pAAAV并进行了测序。序列分析表明,AAAV YZ-1株的基因组为4684bp,两端具有141bp的末端倒置重复序列和Rep蛋白结合位点特征序列,与GenBank中收录的AAAV DA-1株和VR-865株的核苷酸序列同源性分别为95.0%和92.2%。将pAAAV质粒转染CELO病毒感染的鸡胚肝细胞系,获得了感染性AAAV病毒粒子,结果证明克隆的AAAV基因组中存在与病毒复制和包装相关的正确关键序列,可用于重组AAAV载体的构建。  相似文献   

A lost Sorraia maternal lineage found in the Lusitano horse breed     

C. Luís  C. Bastos-Silveira  J. Costa-Ferreira  E.G. Cothran  & M.M. Oom 《Zeitschrift für Tierzüchtung und Züchtungsbiologie》2006,123(6):399-402

A common female founder individual of the Portuguese horse breeds Sorraia and Lusitano was found while conducting research on the variation of the Lusitano mitochondrial DNA lineages in relation to studbook information. We obtained 416‐bp control region sequences from 16 descendents of a female Sorraia founder (Pomba) still represented in the living population of the Lusitano, according to the most recent edition of this breed's studbook. The same haplotype was found for all analysed samples and belongs to the haplogroup described by several authors as having predominantly Iberian, South American and North African haplotypes bringing new insights on the relationship between the Sorraia and the other Iberian breeds. This work illustrates how weak the boundary of breed establishment can be, especially at the same geographical region. Using the same founders in different breeds is surely one of the explanations to frequently shared haplotypes among recent breeds, resulting in a lack of consistency between mtDNA sequences and breeds and/or geographical regions.  相似文献   

中国部分山羊品种线粒体DNA D-loop序列遗传多样性分析   总被引：4，自引：2，他引：4  

刘益平  曹少先  傅泽红  徐敏  刘铁铮 《畜牧兽医学报》2007,38(4):313-320

分析了中国部分本地山羊品种(18个品种200个体)以及在中国饲养的引入品种(4个品种25个体)线粒体DNA控制区(D-loop)的全序列,结合已报道的世界其它地方的山羊(15个品种77个体)以及2只野山羊的线粒体DNA控制区(D-loop)全序列共304个体,研究结果表明:山羊线粒体DNA控制区约为1 212 bp,检测到228个变异位点,203种单倍型,单倍型多样度为0.993±0.001;核苷酸多样度0.018±0.001。中国部分山羊的变异类型主要为类型A和B,而在巴基斯坦山羊中还检测到类型C和D。比较分析结果表明中国山羊遗传多样性和基因交流比中国黄牛品种要高。  相似文献   

口蹄疫病毒WFL株基因组全长cDNA的克隆及序列分析   总被引：1，自引：0，他引：1  

任林柱  王兴龙  李莉  李晓艳  段小宇  梅英武  刘锴  王学理 《中国预防兽医学报》2007,29(1):22-26

根据口蹄疫病毒基因组的结构特点以及GenBank上公布的全序列，用DNAMAN分别设计了涵盖整个基因组序列的3对引物，从接种口蹄疫病毒WFL株的细胞培养液中提取了病毒基因组RNA，采用RT-PCR方法和RACE法分别扩增了3条基因片段，并将扩增片段分别与T载体连接，在体外分别进行了5-半分子和3’半分子的构建。最后将5’半分子和3’半分子连接成基因组全长cDNA分子。经PCR鉴定、酶切鉴定及全长cDNA测序，证实成功构建了口蹄疫病毒wFL株基因组全长eDNA分子。序列分析结果表明，供试口蹄疫病毒基因组全长为8155nt，5＇UTR长1059nt；具有一个大的读码框，其核苷酸长度为6969nt。包括201aa的前导蛋白基因和2122aa的聚合蛋白基因；3＇UTR长127nt，包括34nt的polv（A）。  相似文献   

一株山羊支原体山羊肺炎亚种的分离鉴定与分子特征   总被引：8，自引：0，他引：8  

辛九庆  李媛  张建华  胡守萍  王亮 《中国预防兽医学报》2007,29(4):243-248

从送检的山羊肺炎肺脏中成功分离到一株支原体，经过3次克隆纯化后进行生化试验、电镜观察、PCR及酶切、基因特征鉴定，结果显示分离物SD3属于山羊支原体山羊肺炎亚种成员。将培养物经气管接种2只山羊可引起1只山羊典型发病，体温升高至41．5℃，IgG和IgM抗体效价明显升高，其中IgG抗体变化与临床表现基本同步。剖检发现肺脏发生严重病变，并从中再次分离到该病原体。  相似文献   

香蕉A基因组品种间遗传关系的SSR检测   总被引：3，自引：2，他引：3  

冀小蕊  陈业渊  王静毅  郑丽珊  魏守兴  谢子四  武耀廷 《果树学报》2007,24(6):783-787

应用SSR技术,对32个香蕉A基因组类型品种(系)的遗传关系进行了检测。40对SSR引物在32个品种(系)中分别扩增带数在3～15个,平均每个SSR座位可检测2.99个多态性带;引物的多态信息量(PIC)在0.00～0.88,平均0.62。依据SSR数据计算的品种间遗传距离在0.00%～34.27%,平均12.45%,大多数品种间的遗传变异非常有限,但也存在着遗传差异突出的品种:FHIA25、Yangambi KM5、Pisang Jari Buaya、Rose和皇帝蕉。依据26%的遗传距离,除了FHIA25和Pisang Jari Buaya单独化成1组外,其它30个品种可以分为2组:品种间遗传差异相对较高的组I和品种间遗传差异相对较低的组Ⅱ。Williams与引进的洪都拉斯3号、M931之间,洪都拉斯1号和洪都拉斯2号之间,高脚青芽蕉和高脚顿地雷分别没有区分开来,这可能是同物异名,也可能是同一品种未能分辨的突变体。  相似文献   

长蒴母草黄脉病毒——一个双生病毒的新种     

车海彦  刘先宝  杨旭光  符瑞益  罗大全 《植物病理学报》2007,37(6):578-583

 从采集于海南儋州地区表现黄脉症状的长蒴母草(Lindernia anagallis)上分离到病毒分离物L2, DNA-A全序列分析结果表明, 全长2739个核苷酸(nt)(GenBank登录号:AY795900), 共编码6个ORF, 其中病毒链编码AV1(CP)、AV2, 互补链编码AC1、AC2、AC3、AC4。利用BLAST程序对DNA-A进行分析表明, 与L2 DNA-A有同源关系的病毒均为双生病毒科(Geminiviridae)菜豆金色黄花叶病毒属(Begomovirus)成员。进一步比较发现, L2 DNA-A与我国广东报道的广东番茄曲叶病毒(Tomato leaf curl Guangdong virus, ToLCGuV)(AY602165)全基因组核苷酸序列的同源性最近, 仅为77.0%, 说明L2为Begomovirus中的一个新种, 命名为长蒴母草黄脉病毒(Lindernia anagallis yellow vein virus, LAYVV)。与L2的IR区及各基因编码的氨基酸序列有最高同源性的病毒均来源于亚洲。利用DNA-B特异引物和DNA-β的特异引物, 均未检测到DNA-B和卫星DNA-β的存在。  相似文献