聚合酶链式反应(PCR)技术检测牛奶中金黄色葡萄球菌

根据金黄色葡萄球菌特异性STAA-Aul基因序列,设计、合成了一对引物,运用聚合酶链式反应(PCR)技术从10头患有奶牛乳房炎的奶牛奶样中扩增得到8个大小为420 bp的DNA产物;阴性样品中未扩增到产物。测定了其中2个DNA片段的核酸序列,确定为金黄色葡萄球菌的STAA-Aul基因。     

Multifunctionalised benzoxazinones in the systems Oryza sativa–Echinochloa crus‐galli and Triticum aestivum–Avena fatua as natural‐product‐based herbicide leads

BACKGROUND: Fifteen novel derivatives of D‐DIBOA, including aromatic ring modifications and the addition of side chains in positions C‐2 and N‐4, had previously been synthesised and their phytotoxicity on standard target species (STS) evaluated. This strategy combined steric, electronic, solubility and lipophilicity requirements to achieve the maximum phytotoxic activity. An evaluation of the bioactivity of these compounds on the systems Oryza sativa–Echinochloa crus‐galli and Triticum aestivum–Avena fatua is reported here. RESULTS: All compounds showed inhibition profiles on the two species Echinochloa crus‐galli (L.) Beauv. and Avena fatua L. The most marked effects were caused by 6F‐4Pr‐D‐DIBOA, 6F‐4Val‐D‐DIBOA, 6Cl‐4Pr‐D‐DIBOA and 6Cl‐4Val‐D‐DIBOA. The IC₅₀ values for the systems Echinochloa crus‐galli–Oryza sativa and Avena fatua–Triticum aestivum for all compounds were compared. The compound that showed the greatest selectivity for the system Echinochloa crus‐galli–Oryza sativa was 8Cl‐4Pr‐D‐DIBOA, which was 15 times more selective than the commercial herbicide propanil (Cotanil‐35). With regard to the system Avena fatua–Triticum aestivum, the compounds that showed the highest selectivities were 8Cl‐4Val‐D‐DIBOA and 6F‐4Pr‐D‐DIBOA. The results obtained for 6F‐4Pr‐D‐DIBOA are of great interest because of the high phytotoxicity to Avena fatua (IC₅₀ = 6 µM , r² = 0.9616). CONCLUSION: The in vitro phytotoxicity profiles and selectivities shown by the compounds described here make them candidates for higher‐level studies. 8Cl‐4Pr‐D‐DIBOA for the system Echinochloa crus‐galli‐Oryza sativa and 6F‐4Pr‐D‐DIBOA for Avena fatua‐Triticum aestivum were the most interesting compounds. Copyright © 2010 Society of Chemical Industry     

Deep‐sequencing revealed Citrus bark cracking viroid (CBCVd) as a highly aggressive pathogen on hop

Newly emerging or re‐emerging diseases are a constant and significant threat to agricultural production, so prompt and accurate identification of the causative agents is required for rapid and appropriate disease management. Classical methods of pathogen detection can be successfully supplemented by next‐generation sequencing (NGS), whereby sequence analysis can help in the discovery of new or emerging diseases. In 2007, hop growers in Slovenia reported the appearance of severely stunted hop plants, a phenomenon that spread rapidly within hop gardens and among farms. Classical diagnostic methods were unable to detect a new pathogen; therefore, single step high‐throughput parallel sequencing of total RNA and small RNAs from plants with and without symptoms was employed to identify a novel pathogen. The sequences were assembled de novo and also mapped to reference genomes, resulting in identification of a novel sequence of Citrus bark cracking viroid (CBCVd) in the stunted hop plants. Furthermore, the presence of this novel pathogen on hop was confirmed by RT‐PCR analysis of 59 plants with symptoms from 15 hop gardens, representing the main outbreak locations identified by systematic disease monitoring, and small RNA Illumina sequencing of the bulked RNA sample. The high infectivity of the newly identified CBCVd was also confirmed by biolistic inoculation of two hop cultivars, which developed aggressive symptoms in controlled conditions. This study shows the feasibility of deep sequencing for the identification of causative agents of new diseases in hop and other plants.     

金黄色葡萄球菌FnbpA、ClfA、Ebps抗原表位的串联表达及多克隆抗体的制备

为获得金黄色葡萄球菌特异性抗原蛋白并以此制备多克隆抗体,应用DNAStar软件筛选金黄色葡萄球菌FnbpA、ClfA、Ebps的抗原表位,以柔性氨基酸序列连接后进行全基因合成,将目的基因克隆到pGEX-4T-1原核表达载体,转化至大肠杆菌感受态细胞,诱导表达,采用Sepharose 4B柱亲和层析纯化表达蛋白,制备抗原后对家兔进行皮下多点注射,于第42天采集外周血,获得血清。结果表明,本试验成功构建了金黄色葡萄球菌抗原基因表达载体,并获得了高免血清,经ELISA的方法检测血清效价可达到1∶10000以上,成功制备多克隆抗体,为以后采用免疫学方法快速检测金黄色葡萄球菌奠定了基础。