EFFECTORSEARCH:病原菌基因组中三型分泌系统效应蛋白的预测(英文)

病原菌为了生存和侵入寄主细胞,经过漫长的进化形成了多种可介导大分子转运的分子机制,细菌Ⅲ型分泌系统(T3SS)是其中最重要的一种。T3SS又被称为"细菌注射器",病原菌利用此系统可以将效应蛋白注射到靶细胞中。近年来发现,T3SS不仅存在于在人类、动物、甚至植物的多种致病菌中,在其它环境下的细菌中也存在。本文介绍了一种最新的生物信息学预测工具(EFFECTORSEARCH),可用于在微生物基因组中预测T3SS的基因,并且在本文中着重介绍了其在丁香假单胞菌(Pseudomonas syringae)基因组中T3SS基因的预测结果。

呼吸道病原菌基因检测法在诊断儿童肺炎链球菌感染中的应用价值分析

目的:探讨呼吸道病原菌基因检测法在诊断儿童肺炎链球菌感染中的应用价值。方法:选取梧州市红十字会医院收治的300例初步诊断为细菌性肺炎的患儿作为研究对象。采集这些患儿的支气管肺泡灌洗液或深部痰标本,并对标本进行传统的病原菌培养及呼吸道病原菌基因检测,然后对检测结果进行比较分析。结果:与对这300例患儿进行传统的病原菌培养相比,对其进行呼吸道病原菌基因检测其肺炎链球菌的阳性检出率更高,P<0.05。用呼吸道病原菌基因检测法诊断这300例患儿肺炎链球菌感染的特异度、灵敏度、总符合率、阴性预测值、阳性预测值分别为69.40%、92.37%、83.20%、93.38%、78.67%。本研究中,进行传统的病原菌培养与呼吸道病原菌基因检测诊断儿童肺炎链球菌感染的一致性较好。与对这300例患儿进行传统的病原菌培养相比,对其进行呼吸道病原菌基因检测的用时更短,P<0.05。结论:用呼吸道病原菌基因检测法诊断儿童肺炎链球菌感染的灵敏度、总符合率、阴性预测值等较高,与进行传统病原菌培养的结果一致性较好,且检测用时较短。

基于微流控技术对病原菌基因检测的应用

目的:探讨应用微流控技术对呼吸道病原菌基因检测的效果。方法:通过呼吸道病原菌基因快速检测,标本的DNA提取,经芯片检测。结果:痰标本中,病原菌的检出率70%,其中,46%为单一病原菌感染,54%为两种以上病原菌混合感染。在检出的病原菌中,肺炎链球菌有32例,占46%,其次为肺炎克雷伯菌占19%,鲍曼不动杆菌、铜绿假单胞菌和流感嗜血杆菌占18%,金黄色葡萄球菌占14%,结合分枝杆菌复合群占3%。结论:此方法缩短了病原菌检出的时间,为临床及时给药提供了参考。

呼吸道病原菌基因检测法诊断儿童肺炎链球菌感染的临床价值

目的探讨呼吸道病原菌基因检测法对诊断儿童肺炎链球菌感染的应用价值。方法收集2016年6月—2017年6月在首都儿科研究所附属儿童医院呼吸科住院的初诊细菌性肺炎患儿224例,每位病例采集深部痰或支气管肺泡灌洗液标本,按传统病原菌培养是否分离到肺炎链球菌分为非肺炎链球菌组136例和肺炎链球菌组88例。用呼吸道病原菌核酸检测试剂盒(恒温扩增芯片法)检测2组病例中的病原菌,应用SPSS 17.0统计软件及Kappa检验来对比2种检测方法的检出效能。结果非肺炎链球菌组应用呼吸道病原菌基因检测法检出肺炎链球菌40例,其中深部痰标本检出32例,支气管肺泡灌洗液标本检出8例;肺炎链球菌组应用呼吸道病原菌基因检测法检出肺炎链球菌80例,其中深部痰标本检出52例,支气管肺泡灌洗液标本检出28例。与传统病原学培养对比,呼吸道病原菌基因检测肺炎链球菌的灵敏度、特异度、总符合率、阳性预测值、阴性预测值分别为90.9%、74.4%、80.3%、66.7%、92.3%。2种检测方法对肺炎链球菌的检出率具有较好的一致性(Kappa>0.4)。结论呼吸道病原菌基因检测具有快速、特异、灵敏的优势,与传统的病原学分离培养检验有较高的一致性及总符合率,能为临床医生提供可靠的诊疗依据。

病原菌新基因功能的研究策略

随着高通量测序技术和蛋白组学技术的发展,越来越多的病原菌新基因被发现,研究病原菌新基因有助于更好的防控由该病原菌引起的疾病的传播。本文从生物信息学角度、新基因功能获得与缺失、与新基因编码产物互作蛋白的分析等方面综述了适用于病原菌新基因功能研究的策略,为基因分子生物学等相关领域提供参考。此外,本文还结合常规的新基因研究方法,将芯片技术,2D-DIGE技术渗透入病原菌新基因研究策略中,使研究者能更快捷准确的找出新基因功能研究的切入点,进而制定切实可行的新基因功能的研究方案。

植物抗菌基因工程的研究策略及进展

植物抗菌基因工程是目前植物抗病育种的热点之一 .本文就近几年植物抗菌基因工程中植物抗病基因、病原菌无毒基因、植物防卫基因。

宁波地区致泻性病原菌检测与主要流行株的分子分型研究

目的了解宁波地区致泻性病原菌组成和主要流行株的基因分型,探讨菌株的同源性,为追踪传染源提供依据。方法病原菌检测采用直接与增菌分离相结合的方法;细菌鉴定采用生化筛检和API等法;细菌分型采用诊断血清和PFGE分型。结果本组9 256份样本检出8类16种3 473株致泻性病原菌,检出率为37.52%,以副溶血性弧菌最高,与其他病原菌比差异有统计学意义(P<0.05);血清分型发现副溶血性弧菌的O3群、沙门菌的甲型副伤寒、志贺菌的褔氏志贺菌、致病性大肠埃希菌O119为各病原菌的优势菌;PFGE分型副溶血性弧菌分出19个,伤寒沙门菌分出3个,甲型副伤寒沙门菌分出12个。结论副溶血性弧菌是引起宁波地区感染性腹泻最主要的流行株,PFGE分型能显示菌株间的亲缘关系,PFGE分子分型可分析菌株的来源,为流行病学追踪传染源提供依据。

Construction of Bifidobacterium Infantis/CD Targeting Gene Therapy System

Objective： To construct Bifidobacterium Infantis/CD targeting gene therapy system. Methods： CD gene was amplified from E. Coli K12λ using PCR method, pGEX-1LamdaT plasmid and CD gene were digested with dual restriction endonucleas of EcoR Ⅰ and BamH Ⅰ and two segments of 4.9 kb and 1.3 kb were obtained. T4 DNA ligase was added to these two segments to make a recombinant CD/pGEX-1LamdaT plasmid. Then the recombinant plasmid was transfected into Bifidobacterium Infantis by electroporation. The recombinant plasmid was extracted from the positively transfected Bifidobacterium Infantis and digested with dual restriction endonucleases. Then the size of digested fragments was detected and sequencing of the gene segment inserted in extracted recombinant plasmid was performed according to the method of Sanger dideoxynucleotide triphosphate chain termination. Results： 6.2 kb recombinant plasmid was obtained from the positively transfected bacterial colony of Bifidobacterium Infantis. After being digested with dual restriction endonucleases, two segments of approximate 4.9 kb and 1.3 kb were gained from the extracted recombinant plasmid, which were equal to the size of pGEX-1LamdaT plasmid and CD gene, respectively. The full length and sequence of nucleotide acid of the inserted gene in extracted recombinant plasmid was completely identical to the CD gene. Conclusion： The foreign gene, CD gene was correctly inserted into pGEX-1LambdaT plasmid and transferred into Bifidobacterium Infantis. Bifidobacterium Infantis/CD targeting gene therapy system was successfully constructed.

Molecular Cloning of Early-expressed cDNAs from Rice in Response to Infection by Rice Blast Fungus Magnaporthe grisea

Compatible and incompatible reactions in rice plants ( Oryza sativa L. cv. Shenxianggen No.4) were resulted from inoculation with two different virulent races of rice blast fungus ( Magnaporthe grisea (Hebert) Barr), and thus an effective infecting system was established between rice plants and the rice blast pathogen. Two cDNA clones that showed induced and temporal patterns in expression in the very early stage in response to infection of the fungus were obtained from the plants by use of differential display. Of the two cDNA clones, Fastresp_a was induced to express in both compatible and incompatible interactions although it was expressed earlier in the former reaction. The second one, Fastresp_b , was only expressed in incompatible interaction. Southern blot analysis of the rice genomic DNA indicated that both of the two clones were from genome of the plant. No significant homology to the two genes was found from the rice gene database. This suggested that they were novel genes in rice and may play important roles in rice resistant response to infection of rice blast fungus.

An Indirect ELISA of Classical Swine Fever Virus Based on Quadruple Antigenic Epitope Peptide Expressed in E.coli

In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5%sensitivity and 96.7%specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.

Evaluation of Endophytic Bacteria for Plant Growth Promotion and Pathogen Suppression Traits in Saccharum officinarum

Endophytes are beneficial microbes that are capable of promoting growth, besides protecting colonized plants against plant pathogens. These microbes are of either bacterial, fungal or actinomycetes in plants. In the study, the endophytic bacteria isolated from sugarcane with their characterization related to plant growth promotion and pathogen suppression have been reported. Roots, shoots and leaves of rooted tissue culture plantlets of sugarcane cultivars of 87A298 and 2009A107 were excised aseptically and isolated endophytic bacterial strains. The strains were identified using 16S rRNA gene sequence based homology. Molecular characterization of these strains was carried out for presence of antimicrobial genes. The results showed that the endophytes isolated from sugarcane tissue culture plantlets were of the genera Bacillus （B. amyloliquefaciens, B. subtilis, B. cereus, B. safensis, B. siamensis, B. aryabhattai, B. flexus and B. velezensis） and Paenibacillus pabuli. There were three antimicrobial peptides （AMPs） producing genes of bacilysin, bacillomycin and fengycin in B. amyloliquefaciens （SE1, SE7）, B. siamensis （SE4, SE16）, B. subtilis （SE2, SE3） and B. velezensis （SE15）. The biochemical characterization assays showed that some of these strains could produce hydrogen cyanide （HCN）, protease, cellulase and indole acetic acid （IAA）. Few strains （SE1 and SE4） were phosphate solubilizers, whereas nine isolates were found to be diazotrophs. Most of the bacterial isolates were found antagonistic to Fusarium sacchari, the sugarcane wilt pathogen under in vitro conditions. Overall, the results suggested the scope and potentiality of sugarcane endophytic bacteria, isolated from tissue culture plantlets, in promoting plant growth and suppression of sugarcane pathogen.

Polymorphism of microsatellite sequence within signal transduction coding genes in phytopathogenic fungus, Magnaporthe grisea

Sixteen polymorphic microsatellite markers suitable for population genetic structure analysis and signal transduction coding genes variation measurement were developed for rice blast fungus, Magnaporthe grisea. Polymorphism was evaluated by using forty-six isolates collected from diverse geographical locations （including japonica grown zone, indica grown zone） and rice varieties. Preliminary results indicated that each locus resolved multiple alleles ranging from three to fourteen. The results showed that these SSR-containing genes are also polymorphic in the nature population.

Recent advances in the molecular genetics of resin biosynthesis and genetic engineering strategies to improve defenses in conifers

Since the first terpenoid synthase cDNA was obtained by the reverse genetic approach from grand fir, great progress in the molecular genetics of terpenoid formation has been made with angiosperms and genes encoding a monoterpene synthase, a sesquiterpene synthase, and a diterpene synthase. Tree killing bark beetles and their vectored fungal pathogens are the most destructive agents of conifer forests worldwide. Conifers defend against attack by the constitutive and inducible production of oleoresin that accumulates at the wound site to kill invaders and both flush and seal the injury. Although toxic to the bark beetle and fungal pathogen, oleoresin also plays a central role in the chemical ecology of these boring insects. Recent advances in the molecular genetics of terpenoid biosynthesis provide evidence for the evolutionary origins of oleoresin and permit consideration of genetic engineering strategies to improve conifer defenses as a component of modern forest biotechnology. This review described enzymes of resin biosynthesis, structural feathers of genes genomic intron and exon organization, pathway organization and evolution, resin production and accumulation, interactions between conifer and bark beetle, and engineering strategies to improve conifer defenses.

Investigation on detection of Haemophilus ducreyi by Polymerase Chain Reaction

Objective:To investigate the application of polymerase chain reaction (PCR) detection of Haemophilus ducreyi in clinical diagnosis of chancroid. Methods: Nucleotide sequences of 16srRNA gene specific for H. dureyi were used to develop primer sets for amplification of two strains. The amplified products were tested via PCR and sequenced by electrophoresis in a 1.5% gel.These products were compared with those of heterogeneous species or related bacteria to test the specificity of the PCR assay. PCR amplification with different concentrations of H.ducreyi was performed to test its sensitivity. Results: PCR amplification of two strains of H. ducreyi produced a single band of expected 438bp length. The sequence was identified with genomic DNA. None of the other 19 reference species amplified under the same conditions gave this result. The highest sensitivity of PCR assay in the present test was 10ng/L. Conclusions: PCR assay for detection of H. ducreyi is a rapid, specific, and sensitive detection method. If laboratory conditions are strictly controlled, PCR assay is a potentially useful laboratory test for H. ducreyi infection diagnosis.

Isolation and characterization of a crude oil degrading bacteria from formation water:comparative genomic analysis of environmental Ochrobactrum intermedium isolate versus clinical strains

In this study, we isolated an environmental clone of Ochrobactrum intermedium, strain 2745-2, from the formation water of Changqing oilfield in Shanxi, China, which can degrade crude oil. Strain 2745-2 is aerobic and rod-shaped with optimum growth at 42 ℃ and pH 5.5. We sequenced the genome and found a single chromosome of 4800175 bp, with a G＋C content of 57.63%. Sixty RNAs and 4737 protein-coding genes were identified： many of the genes are responsible for the degradation, emulsification, and metabolizing of crude oil. A comparative genomic analysis with related clinical strains （M86, 229E, and LMG3301T） showed that genes involved in virulence, disease, defense, phages, prophages, transposable elements, plasmids, and antibiotic resistance are also present in strain 2745-2.