斑点叉尾鮰肠败血症PCR诊断方法的建立

为了建立快速、敏感的聚合酶链式反应(PCR)诊断斑点叉尾鮰Ictalurus punctatus的肠败血症技术,作者根据NCB I公布的鮰爱德华氏细菌序列,设计了一对特异性诊断引物CM3/CM4,对鮰爱德华氏细菌特异基因片段进行PCR扩增试验,PCR反应条件的优化以及不同检测材料的比较,同时测试了该方法的特异性和敏感性,并对广西4个市县临床样品进行了检测。结果表明:用该引物可以扩增到与预计大小相符的276 bp鮰爱德华氏菌特异性片段;PCR反应与Ⅰ型荧光假单胞菌、点状产气单胞菌点状亚种、鳗弧菌、温和气单胞菌、肠型点状产气单胞菌、柱状黄杆菌、嗜水气单胞菌、河弧菌、迟缓爱德华氏菌及海豚链球菌无交叉反应;用该PCR法可以检测出病鱼脑、肝、肾及脾中的病原菌,检测的最低细菌数为12个,且病鱼内脏组织、菌落及菌液均可直接用于该PCR扩增;临床菌株检测结果与基于菌株16S rRNA基因序列系统进化分析和生化鉴定结果一致。因此,本试验中所建立的PCR检测方法在斑点叉尾鮰肠败血症的诊断和防治方面具有较好的应用前景。     

养殖大菱鲆感染迟缓爱德华氏菌的分离、毒力基因及ERIC-PCR分析

为了调查辽宁省葫芦岛市养殖患病大菱鲆Scophthalmus maximus病原菌感染情况,选用迟缓爱德华氏菌Edwardsiella tarda特异性引物对分离菌株进行PCR鉴定,共分离得到22株迟缓爱德华氏菌。结果表明:迟缓爱德华氏菌毒力基因的PCR扩增显示,22株菌均含有kat B、fim A、gad B、esa V等基因;人工感染试验表明,22株迟缓爱德华氏菌感染并致大菱鲆累积死亡率均为100%;ERIC-PCR结果显示,迟缓爱德华氏菌模式菌株(ATCC15947)与分离株可分为2个基因型,分别标记为Ⅰ和Ⅱ型,其中22株迟缓爱德华氏菌分离株均为Ⅱ型,与ATCC15947菌株为Ⅰ型明显不同。本研究结果为养殖大菱鲆迟缓爱德华氏菌感染的防控提供了依据。     

鸡抗迟缓爱德华氏菌卵黄抗体的应用研究

从全身出血合并腹水的养殖大菱鲆Scophthalmus maximus体内分离出1株细菌,经感染试验证实其病原性,并鉴定该菌为迟缓爱德华氏菌Edwardsiella tarda。采用该菌制成全菌油乳性灭活疫苗,分4次给蛋鸡进行免疫注射,第2次加强免疫后开始收集鸡蛋,制备卵黄抗体(IgY)和其水溶性组分(W SF),并用试管凝集法测定其效价。取效价达到1∶64的鸡蛋提取卵黄抗体,分别采用口服和腹腔注射两种方式给大菱鲆进行免疫:配成含W SF 1 mL/g的饵料,给鱼连续投喂15 d;用含蛋白含量0.19 mg/mL的纯化IgY,于第1、3、14 d对大菱鲆进行腹腔注射,剂量为0.1 mL尾/。免疫后分别用注射和划伤浸泡两种方法攻毒。结果表明,口服特异性卵黄抗体对大菱鲆的免疫保护率分别为83.3%和100%,而注射免疫没有保护作用。     

铜绿假单胞菌mexA基因原核表达载体的构建

目的构建mexA基因的原核载体,为进一步表达MexA蛋白奠定基础。方法从临床分离多重耐药的铜绿假单胞菌抽提DNA,经PCR扩增出mexA1基因与PUC18克隆载体连接,PCR、酶切及测序鉴定,再以PUC/mexA1质粒为模板PCR扩增mexA目的基因,克隆至质粒pQE30中,构建表达质粒pQE30/mexA,构建的表达质粒pQE30/mexA转化大肠杆菌M15,培养后提取纯化重组质粒pQE30/mexA酶切鉴定。结果获得长约1.5 kbPCR mexA1产物,酶切结果显示所构建的重组质粒PUC/mexA1已成功地克隆了mexA1（1.5 kb）基因,序列分析结果与PAO1/mexA1序列相同,构建的表达质粒pQE30/mexA酶切鉴定,与插入pQE30的目的基因mexA（1.2 kb）片段相符。结论含mexA（1.2 kb）基因的原核表达载体构建成功,为进一步表达MexA蛋白奠定了基础。     

玉米弯孢叶斑病菌ATMT突变株构建及致病力分析

利用农杆菌介导的基因转化(Agrobacterium-mediated transformation,ATMT)技术,转化玉米弯孢叶斑病菌(Curvularia lunata),共获得转化子1 454个。对其中菌落形态、生长速率等性状有显著变化的8个突变株进行分析。通过离体叶片接种进行筛选,发现4个突变株致病性降低,2个突变株致病性增强。通过测定生长速率、产孢量及细胞壁降解酶活性发现,其中致病性减弱的突变株,其产孢量均有所下降甚至不产孢,突变株的PG酶活性普遍增强,但Cx酶活性没有明显变化,而2株强致病性突变株的Cx酶活性明显增强。     

GPR126条件性基因敲除载体的快速构建

基因敲除小鼠模型是在动物体内研究基因功能的重要和可靠的手段之一,而构建用于同源重组的基因敲除载体是构建基因敲除小鼠模型的关键一步。条件性基因敲除技术将针对靶基因的修饰作用限制在小鼠的特定发育时期或特定类型的组织细胞中,通过加入具有位点特异性的重组系统,该修饰作用在时间和空间上均处于可调控的状态;条件性基因敲除技术既可以避免某些具有重要功能基因的敲除所带来的早期胚胎致死问题,还可用于精确分析某些多效基因在不同组织或不同发育阶段的特定功能差异。通过合理利用改良的Red重组系统,将两个LoxP位点快速准确地插入到了目标位置,实现了GPR126条件性基因敲除载体的快速构建,为后续的构建GPR126基因敲除小鼠模型、在动物体内研究基因的功能奠定了基础。     

GSTT1基因缺失、多态性与肺癌易感性的研究

目的:探讨GSTT1基因缺失、多态性与肺癌易感性的关系及其临床意义。方法:选取2016年4月-2017年8月在本院接受治疗的84例肺癌患者为研究组,另选取100例健康人群作为对照组,采用多重PCR技术检测研究对象的GSTT1基因缺陷情况。比较两组GSTT1基因缺陷情况,比较两组中吸烟与不吸烟者的GSTT1基因缺陷情况,比较鳞癌患者与对照组的GSTT1基因缺陷情况。结果:研究组GSTT1(-)型基因缺失情况多于对照组(P<0.05);研究组吸烟者中GSTT1(-)情况多于对照组(P<0.05);鳞癌患者中GSTT1(-)型基因缺失情况多于对照组(P<0.05)。结论:GSTT1基因缺失、多态性与肺癌易感性息息相关,GSTT1(-)基因型有增加患肺癌的可能性,且与吸烟患者存在交互作用,进一步的研究将更好地理解目标基因如何影响肺癌风险,对于肺癌的预防和治疗有重要意义。     

丙型肝炎病毒JFH1适应性突变株全长cDNA的构建

目的构建丙型肝炎病毒JFH1全长c DNA克隆,获取突变位点信息。方法应用长片段RT长片段RT-PCR技术对不同时期传代的JFH1病毒株进行分段扩增。通过共同酶切位点连接成9.7Kb从DNA片段,与p Blue Script2ks(+)形成克隆载体,进一步测序分析。结果获取丙型肝炎病毒JFH1系列适应性突变株全长c DNA模板,共发现23个单核苷酸突变。结论丙型肝炎病毒JFH1系列存在单核苷酸突变位点,为进一步了解HCV JFH1适应性突变位点功能奠定基础。     

植物乳杆菌KLDS1.0391 luxS基因敲除载体的构建与应用

构建植物乳杆菌KLDS1.0391的luxS基因敲除载体并实现基因敲除。以植物乳杆菌KLDS1.0391的基因组为模板,采用PCR方法扩增luxS侧翼序列luxSL、luxSR并分别克隆至pMD19-T simple中,鉴定正确后酶切,与敲除载体pNZ5319连接,鉴定正确后转入大肠杆菌DH5α中保存。采用电转化方法将敲除载体导入植物乳杆菌KLDS1.0391,构建luxS基因敲除菌株。结果表明,植物乳杆菌luxS基因敲除载体pNZ5319-luxS构建成功,测序结果证实该菌中存在2个luxS基因且成功敲除1个。所得结果为进一步研究luxS基因在植物乳杆菌KLDS1.0391的代谢和群体感应中的重要作用奠定了基础。     

Kinetics of a hydrogen-oxidizing, perchlorate-reducing bacterium

This paper provides the first kinetic parameters for a hydrogen-oxidizing perchlorate-reducing bacterium (PCRB), Dechloromonas sp. PC1. The qmax for perchlorate and chlorate were 3.1 and 6.3 mg/mgDW-day, respectively. The K for perchlorate was 0.14 mg/L, an order of magnitude lower than reported for other PCRB. The yields Y on perchlorate and chlorate were 0.23 and 0.22 mgDW/mg, respectively, and the decay constant b was 0.055/day. The growth-threshold, Smin, for perchlorate was 14 microg/L, suggesting that perchlorate cannot be reduced below this level when perchlorate is the primary electron-acceptor, although it may be possible when oxygen or nitrate is the primary acceptor. Chlorate accumulated at maximum concentrations of 0.6-4.3 mg/L in batch tests with initial perchlorate concentrations ranging from 100 to 600 mg/L. Furthermore, 50 mg/L chlorate inhibited perchlorate reduction with perchlorate at 100 mg/L. This is the first report of chlorate accumulation and inhibition for a pure culture of PCRB. These Chlorate effects are consistent with competitive inhibition between perchlorate and chlorate for the (per)chlorate reductase enzyme.     

Kinetics of a chlorate-accumulating, perchlorate-reducing bacterium

Kinetics parameters for perchlorate and chlorate reduction were determined for Dechlorosoma sp. HCAP-C, also known as Dechlorosoma sp. PCC, a novel perchlorate-reducing bacterium (PCRB) that accumulates significant amounts of chlorate during perchlorate reduction. This is the first report of such behavior, and we hypothesized the perchlorate reduction kinetics would be markedly different from other PCRB. In batch tests with initial perchlorate concentrations ranging from 200 to around 1400 mg/L, maximum chlorate accumulation ranged from 41 to 279 mg/L, and were consistently around 20% of the initial perchlorate concentration. For perchlorate, parameters were determined using a competitive inhibition model. The maximum specific substrate degradation rate qmaxP was 11.5mgClO4-/mgdry weight (DW)-d, and the half-maximum rate constant KP was 193 mgClO4-/L. For chlorate, the qmaxC was 8.3 mgClO3-/mgDW-d and the KC was 58.3 mgClO3-/L. The high KP values relative to conventional PCRB, values suggests that HCAP-C does not play a significant role at low perchlorate concentrations. However, the relatively high qmaxP, and the potential for syntrophic relationships with chlorate-reducing bacteria that relieve the effects of chlorate inhibition, suggest that HCAP-C could play a significant role at high perchlorate concentrations.     

软骨发育不全症与成纤维细胞生长因子受体-3致病基因

软骨发育不全(ACH)是一种由于软骨内骨化缺陷所致的发育异常,临床上以四肢短、躯干相对正常、巨头、脊柱胸腰段后凸、椎管狭窄为特征,是成纤维细胞生长因子受体-3(FGFR3)基因突变所致疾病。本文就ACH致病基因FGFR3的结构、功能、致病机制、产前诊断的有关进展作一综述。     

氨氮降解菌的分离、鉴定及降解效果初步研究

以(NH4)2SO4为唯一氮源的培养基中,从活性污泥中分离筛选出8株具有氨氮降解能力的菌株,根据各菌株之间降解率及生长情况的比较,从中筛选出1株对氨氮降解效果较为明显的菌株NX3,经形态学和生理特性初步鉴定其为芽孢杆菌属(Bacillus),分别测定了在不同的氨氮初始浓度、pH值、温度下菌株NX3对培养基中氨氮的降解效果,实验结果表明在初始氨氮质量浓度300mg/L、pH值7.0、温度30℃时,该菌株对氨氮降解效果较好,降解率为45.53%。     

Removal of fish pathogenic bacteria in biological sand filters

Documentation is required to evaluate the use of infiltration systems as an alternative method for removal of fish pathogenic bacteria in wastewater from fish-farms. This study was performed to investigate the removal of bacterial fish pathogens in biological sand filters. A second aim of the study was to evaluate the bacteria used in the study in order to find a suitable model organism for future experiments. Low-strength wastewater from an inland freshwater salmonid farm was intermittently loaded (70 mm/day in 24 doses) to filter columns containing either fine sand (d(10)=0.25) or coarse sand (d(10)=0.86). After a wastewater loading period of 10 weeks, separate sand columns were seeded with Yersinia ruckeri, Pseudomonas fluorescens, Aeromonas hydrophila and Aeromonas salmonicida subsp. salmonicida, respectively, for a period of 30 days. All the bacteria showed the same removal performance during the experiment, with a significantly lower removal in the beginning of the experiment (day 1-7) compared to mid- and late-phase (day 12-30). In mid- and late-phase the removal stabilized at a high level (>99.9%) for all the bacteria. The hydrophobic cell surface properties of the Aeromonads were higher than Ps. fluorescens and Y. ruckeri. This can possibly explain the significantly higher (P<0.05) removal efficiencies seen for A. hydrophila and A. salmonicida subsp. salmonicida compared to Y. ruckeri and Ps. fluorescens. Results were promising with regard to the use of low-cost infiltration systems as an alternative disinfection method for fish-farm wastewater. Following the criteria for a suitable model organism (removal efficiency, detection in filter effluent and die-off in storage tanks), Y. ruckeri was found to be a feasible model organism for use in future experiments.     

Characterization of degradation process of cyanobacterial hepatotoxins by a gram-negative aerobic bacterium

A bacterium termed 7CY, capable of decomposing cyanobacterial toxins, was isolated from surface water sample of Lake Suwa and degradation of microcystin-RR and nodularin-Har was investigated. The isolated 7CY was a gram-negative, aerobic bacillus, and a member of a genus Sphingomonas. The strain degraded microcystin-LY, -LW, and -LF completely as well as microcystin-LR within 4 days after their addition (6 microg/ml) whereas degradation of nodularin-Har did not occur at all during experiment. On the contrary, the strain was capable of degrading nodularin-Har in the presence of microcystin-RR and both toxins were completely decomposed within 6 days. The strain scarcely degraded nodularin-Har in the presence of microcystin-RR when glucose and ammonium chloride were added to the medium. The degradation of nodularin-Har did not occur in the medium from which bacterial cells had been removed after degradation of microcystin-RR. Furthermore, when microcystin-RR and nodularin-Har were added to the cytoplasm fraction of 7CY cells, microcystin-RR was rapidly degraded within 18 h, but nodularin-Har was not. The strain 7CY may require an enzyme(s) induced during the degradation of microcystin-RR in order to utilize nodularin-Har as nutrition.     

Thermal inactivation of water-borne pathogenic and indicator bacteria at sub-boiling temperatures

The use of harvested rainwater in domestic hot water systems can result in optimised environmental and economic benefits to urban water cycle management, however, the water quality and health risks of such a scenario have not been adequately investigated. Thermal inactivation analyses were carried out on eight species of non-spore-forming bacteria in a water medium at temperatures relevant to domestic hot water systems (55-65 degrees C), and susceptibilities to heat stress were compared using D-values. The D-value was defined as the time required to reduce a bacterial population by 90% or 1 log reduction. The results found that both tested strains of Enterococcus faecalis were the most heat resistant of the bacteria studied, followed by the pathogens Shigella sonnei biotype A and Escherichia coli O157:H7, and the non-pathogenic E. coli O3:H6. Pseudomonas aeruginosa was found to be less resistant to heat, while Salmonella typhimurium, Serratia marcescens, Klebsiella pneumoniae and Aeromonas hydrophila displayed minimal heat resistance capacities. At 65 degrees C, little thermal resistance was demonstrated by any species, with log reductions in concentration occurring within seconds. The results of this study suggested that the temperature range from 55 to 65 degrees C was critical for effective elimination of enteric/pathogenic bacterial components and supported the thesis that hot water systems should operate at a minimum of 60 degrees C.     

Isolation and identification of a novel microcystin-degrading bacterium from a biological sand filter

A novel bacterium capable of degrading two microcystin analogues, microcystin-LR and -LA (MCLR and MCLA), was isolated from a biological sand filter which was previously shown to effectively remove these toxins from source waters. Based on phylogenetic analysis of the 16S rRNA gene sequence, the isolated organism, LH21, most likely belonged to the genus Sphingopyxis and of the previously cultured species clustered with Sphingopyxis witflariensis. Using polymerase chain reaction (PCR), isolate LH21 was shown to contain homologues to each of the four genes, mlrA, mlrB, mlrC and mlrD previously associated with the degradation of MCLR by Sphingomonas sp. ACM-3962. Isolate LH21 was able to effectively degrade MCLR and MCLA in batch experiments under environmentally relevant conditions, with complete removal observed within 5 h after re-exposure of the toxins.     

高温菌发酵甘露醇高效产乙醇的代谢途径研究

甘露醇是大型褐藻的主要成分之一,在工业生产海藻酸的过程中,甘露醇成为副产物被废弃。筛选到一株高温厌氧芽孢杆菌Rx1,利用甘露醇发酵,乙醇得率高达1.8 mol乙醇/mol甘露醇,为理论产值的90.5%,是发酵葡萄糖乙醇得率的1.7倍,还发现菌株发酵甘露醇的初期会产生微量的乙酸,随后又被立即消耗。通过添加外源乙酸对Rx1代谢特性进行研究,结果外源乙酸不仅能促进底物的利用,还发现Rx1存在转化乙酸生成乙醇的途径,而该途径关键酶的合成需要甘露醇的诱导。