正交设计优化假俭草SRAP-PCR反应体系及引物筛选   总被引：16，自引：7，他引：9  

郑轶琦  王志勇  郭海林  薛丹丹  刘建秀 《草业学报》2008,17(4)

以假俭草叶片DNA为模板,采用正交试验设计,以Mg2 、dNTP、引物和Taq DNA聚合酶4种因素3个水平,对假俭草SRAP反应体系进行研究,并比较了不同浓度模板DNA对扩增效果的影响,建立了假俭草的SRAP最佳反应体系.结果表明,假俭草SRAP-PCR最佳反应体系为:2 μL 10譖CR buffer、60 ng模板DNA、Mg2 1.50 mmol/L、dNTP 260 μmol/L、引物0.25 μmol/L、Taq DNA聚合酶0.5 U,总体积为20 μL.各因素对扩增反应结果均有不同影响,其中以Mg2 浓度影响最大,Taq DNA聚合酶的影响最小.运用该体系对4份假俭草种源进行验证,证明该体系稳定可靠,并从45个SRAP引物组合中筛选出扩增条带清晰、多态性丰富的26个引物组合.这一体系的建立及多态性引物组合的筛选为今后利用SRAP标记技术进行假俭草分子遗传学研究提供了科学依据.  相似文献   

重组马立克病毒通用转移载体的构建及初步应用     

刘召明  张丽萍  王秀丽  赵冬凤  刘新文  李明义 《中国动物检疫》2008,25(1):26-28

本实验以独立启动子控制的增强型绿色荧光基因(GFP)作为报告基因,同时将CMV启动子及其多克隆位点与之连接,构成外源基因表达盒,插入到马立克病毒(MDV)复制非必需区基因(短独特区US2等)构成的同源臂中,构建成重组马立克病毒的通用载体。鉴定正确后,将转移载体与提取的MDV基因组共转染鸡胚成纤维细胞(CEF),同源重组获得具有感染性的重组病毒,待病毒蚀斑出现后,荧光显微镜下观察,可见到明显的绿色荧光病毒蚀斑,经三次筛选,初步分离到重组病毒。结果表明,转移载体与MDV基因组共转染可获得感染性病毒,US2基因可作为重组病毒构建中的外源基因插入位点,证实通用转移载体的构建是可行的,为重组马立克病毒新型疫苗的研究奠定物质基础。  相似文献   

菊欧文氏菌分子检测技术的研究   总被引：1，自引：0，他引：1  

刘鹏  黄国明  刘勇  崔铁军 《植物病理学报》2007,37(6):584-587

 蝴蝶兰细菌性软腐病对蝴蝶兰的生长危害严重, Erwinia chrysanthemi(菊欧文氏菌)、Erwinia carotovora subsp. carotovora(胡萝卜软腐欧文氏菌胡萝卜软腐亚种)是引起蝴蝶兰软腐病的主要病原细菌, 其中E.chrysanthemi被列入我国三类检疫性有害生物。本文对菊欧文氏菌分子检测技术进行了研究, 设计出针对该病原细菌的特异性引物, 应用实时荧光PCR方法检测样品中存在的菊欧文氏菌, 检测灵敏度达到10²cfu/mL。  相似文献   

不同引物RT-PCR方法检测猪繁殖与呼吸综合征病毒的比较研究   总被引：9，自引：5，他引：9  

邓雨修  李玉峰  姜平  蒋文明  汤景元 《畜牧与兽医》2006,38(4):8-11

用3对分别针对猪繁殖与呼吸综合征病毒(PRRSV)的ORF7、ORF5和ORF5的PCR引物N1/N2、AdGP5.1/AdGP5.2和RFLP5.1/RFLP5.2进行RT-PCR,检测PRRSV,从其敏感性、特异性和临床样品检出率等方面进行比较,在此基础上进一步建立一步法RT-PCR检测方法。结果显示:3对引物对PRRSV均有很高的特异性;应用N1/N2引物病毒最低检测量为7.9 TC ID50,而AdGP5.1/AdGP5.2引物和RFLP5.1/RFLP5.2引物PCR最低检测量为79 TC ID50;运用N1/N2、AdGP5.1/AdGP5.2和RFLP5.1/RFLP5.2引物分别进行RT-PCR扩增检测临床样品,PRRSV检出率分别为28/48、27/48和25/48,且用AdGP5.1/AdGP5.2和RFLP5.1/RFLP5.2引物检测的阳性样品,用N1/N2引物检测也都呈阳性。运用N1/N2引物,通过一步法RT-PCR成功地从PRRSV S1株中扩增出374 bp的目的基因片段。结果表明,用N1/N2引物扩增PRRSV目的基因,其敏感性和临床样品检出率更高,更适合临床样品PRRSV的检测。  相似文献   

Detection of Foot and Mouth Disease Virus by RT-PCR and Microplate Hydridization Assay Using Inactivated Viral Antigens     

Barlic-Maganja D  Grom J  Toplak I  Hostnik P 《Veterinary research communications》2004,28(2):149-158

A single step RT-PCR was tested for detection of foot and mouth disease virus (FMDV) and immunoenzymatic determination of amplified products in a microplate hybridization assay. Inactivated reference strains (ELISA antigen) of all seven serotypes were used to optimize the test. Oligonucleotide primers were selected from two different genomic regions coding for RNA polymerase and VP1 protein, respectively. The RT-PCR used to amplify the polymerase gene specific RNA detected FMDV strains A, C, O, Asial and SAT1, and the identity of the fragments obtained was confirmed with a specific internal biotin-labelled capture probe. For the amplification of the VP1 genome region, two sets of oligonucleotide primers were used. One primer pair was successfully applied for the detection of serotypes A, C, O and Asial and a second one for serotypes SAT1, SAT2, SAT3. The specific probe enabled the detection of all the amplified products in a PCR ELISA test. By comparison with antigen ELISA, the PCR ELISA method allowed the detection of smaller amounts of FMDV in the inactivated material examined. The application of molecular diagnostic methods to inactivated antigens offers a good alternative procedure for developing and optimizing a sensitive method for detection of FMDV in laboratories that are not allowed to work with viable FMDV.  相似文献   

Reliable detection of the fungal pathogenfusarium oxysporum f.sp.albedinis, causal agent of bayoud disease of date palm, using molecular techniques     

Stanley Freeman  Marcel Maymon 《Phytoparasitica》2000,28(4):341-348

Bayoud, caused by the soilborne fungusFusarium oxysporum f.sp.albedinis (FOA), is the most serious disease of date palm. Since the disease is located in the North African countries of Morocco and Algeria, and advancing steadily eastwards, the ultimate goal is to prevent spread of the pathogen to other date-growing areas in the region and farther afield. Molecular diagnostic techniques have been developed for detection of FOA. In view of the fact that the fungus does not exist in Israel, DNA of FOA was obtained to determine the reliability of these methods for diagnostic purposes. Random amplified polymorphic DNA was not reliable enough for differentiation between FOA and various pathogenic and saprophyticFusarium isolates. However, the polymerase chain reaction utilizing FOA-specific primers was accurate and enabled amplification of a unique band specific to FOA DNA alone, and not that of the other tested pathogenic and saprophyticFusaria. The availability of a rapid and reliable diagnostic tool for detection of FOA will enable the Plant Protection and Inspection Services of the Israel Ministry of Agriculture to test date palm tissue for the presence of the pathogen. Contribution no. 513/00 from the Inst. of Plant Protection, ARO, The Volcani Center, Bet Dagan, Israel.  相似文献   

Usefulness of single copy genes containing introns in Phytophthora for the development of detection tools for the regulated species P. ramorum and P. fragariae     

Renaud Ioos  Lise Laugustin  Nathalie Schenck  Sylvie Rose  Claude Husson  Pascal Frey 《European journal of plant pathology / European Foundation for Plant Pathology》2006,116(2):171-176

Introns are generally highly polymorphic regions within genes and were proven to be of great interest for discriminating among phylogenetically-close Phytophthora species. Phytophthora ramorum and P. fragariae are considered as quarantine pathogens by the European Union and accurate detection tools are therefore necessary for their monitoring. From introns located in different single copy genes (GPA1, RAS-like, and TRP1), we developed a series of PCR primers specific to P. ramorum and P. fragariae. The specificity of these primers was successfully checked with a wide collection of Phytophthora isolates and a protocol was developed to detect both pathogens directly in infected plant tissues. These genes should be of particular interest for the development of additional species-specific detection tools within the Phytophthora genus.  相似文献   

Rapid bidirectional allele-specific PCR identification for triazine resistance in higher plants     

Tian X  Darmency H 《Pest management science》2006,62(6):531-536

Triazine resistance is reported to be due to chloroplast herbicide target insensitivity in most species, and this is most often caused by a Ser(264)-Gly mutation at the D1 protein. In order to ascertain whether this mutation is really predominant amongst resistant plants, and also for gene flow studies, a rapid test is needed that allows the testing of large quantities of plants. Here a bidirectional allele-specific PCR (polymerase chain reaction) identification is proposed. The designed primers were shown to be universal in the three grass and three broadleaf species examined.  相似文献   

创新植保推广模式促进植保技术的普及     

王新学  李兰蕊  李雪丽 《河北农业科学》2014,(1):86-89

为适应现代植保要求,加快植保新技术的普及和应用,高阳县植保植检站通过建立植保专防队、农民培训、实施农业项目、采用现代信息技术等多种植保推广新模式的综合应用,提高了植保新技术的普及率和到位率,创新了植保推广模式,促进了植保技术的推广。  相似文献   

基于GIS和USLE的九龙江流域土壤侵蚀量预测研究   总被引：63，自引：1，他引：63  

黄金良洪华生  张珞平杜鹏飞 《水土保持学报》2004,18(5):75-79

探讨了GIS和USLE相结合预测南方中等尺度流域土壤侵蚀量、标识流域土壤侵蚀严重区域。运用GIS建立九龙江流域基础地理数据库，利用ARC／INFO的栅格数据空间分析功能，根据USLE土壤侵蚀预测模型对数据库进行图形运算，预测了九龙江流域的土壤侵蚀量。结果表明，流域的年均侵蚀模数为2730．3t／km^2，侵蚀强度属中度。占流域面积85．72％的区域土壤侵蚀强度在中度以下。这一区域对流域土壤侵蚀量的贡献率为58．26％，而流域41．74％的侵蚀泥沙来自于占流域面积14．28％的强度以上侵蚀区域。在流域侵蚀强度的空间分布上，8个子流域属中度侵蚀区，其中船场溪、花山溪和雁石溪三个子流域侵蚀强度较大；6个子流域属轻度侵蚀区，其中漳州平原的龙海和浦南两子流域侵蚀强度最弱。  相似文献