溶藻弧菌溶血素基因反向PCR克隆及其原核表达

溶藻弧菌的胞外产物(ECP)在其致病过程中发挥重要作用,已经观察到溶藻弧菌ECP所致的溶血现象,关于溶藻弧菌溶血素的种类和其对溶藻弧菌的致病性贡献的报道非常稀少。本文利用已经报道的多种弧菌溶血素基因序列设计通用引物,检测溶血素基因在溶藻弧菌中的分布,发现96个菌株中有74株扩增出溶血素基因(vah)片段。对致病株ZJ0451的 vah片段测序。根据已测得的片段序列设计引物,通过反向PCR扩增及后续克隆测序获得全长vah基因及部分侧翼序列。经比对证实溶藻弧菌vah与多种弧菌的TLH类溶血素高度相似,且其肽链N端有信号肽。利用pET32a载体构建了两种包含不同长度融合标签的vah表达载体,并均在大肠杆菌BL21(DE3)中获得可溶性表达。在26℃的诱导温度下,9h时vah表达量达到相对最大。在原核表达系统中,vah蛋白信号肽可以被切除。     

基于单链DNA浓度法的哈维氏弧菌核酸适配体亲和特异性研究

本研究采用核酸适配体的单链DNA浓度来表征其亲和力,通过测定核酸适配体与靶细菌哈维氏弧菌(Vibrio harveyi)结合后ssDNA的浓度,来研究该核酸适配体的亲和特异性和亲和常数,并通过荧光显微镜法对其亲和特异性进行验证。结果显示,采用单链DNA浓度法测得该核酸适配体对靶细菌哈维氏弧菌的亲和力是非目标菌的15.2倍以上;荧光显微镜直接观察发现只有靶细菌能较好结合有荧光标记的核酸适配体,并呈现明显荧光;荧光阻断法发现靶细菌和非靶细菌都未呈现明显荧光,证明了该适配体有较好的亲和特异性,也进一步验证了单链DNA法的测定结果。在亲和常数的测定方面,利用单链DNA浓度法测得该核酸适配体的亲和常数K_d=(33.70±7.83) nmol/L,相应的拟合系数为R~2=0.960,有较好的准确性和可靠性,说明采用单链DNA浓度法来测定适配体的亲和力和亲和常数是可行的。     

应用RD-PCR技术构建溶藻弧菌的cDNA文库

应用限制性显示PCR(RD-PCR)技术构建溶藻弧菌poly(A)化mRNA的cDNA文库,筛选,收集巧妙地解决了由于细菌mRNA poly(A)化位点的高度多态性、用常规方法构建原核cDNA文库所遇到的文库大量重复冗余的难题。并进行筛选、收集cDNA片段进行测序,根据测序结果进行生物信息学分析和结果验证。实验结果表明,我们成功地构建了溶藻弧菌poly (A)化mRNA的cDNA文库,获得一百多个基因片段并对其中53个基因片段进行测序分析,获得了如细菌Ⅲ型分泌系统易位蛋白、趋药性传感器、类丝氨酸蛋白酶等毒力相关因子的基因片断。并且在一定程度上证实了poly(A)化在细菌中不是个别和偶然的现象。实验结果说明了我们所构建的溶藻弧菌poly(A)化mRNA的cDNA文库质量高,文库基因片段的重复性低,筛选基因片段效率高、目的性强。本文并探讨了限制性显示PCR技术在细菌poly(A)化mRNA的cDNA1文库构建中的应用价值。     

基于一种新基因的溶藻弧菌毒力菌株检测方法的建立

根据溶藻弧菌毒力菌株HN08155的1个新的特异性基因序列设计了1对特异性引物SDRHf和SDRHr,扩增产物大小为217 bp;以此引物对为基础成功建立了与HN08155具有相似遗传型的溶藻弧菌毒力菌株PCR快速分子检测试剂盒.该试剂盒具有快速、灵敏度高、特异性强等优点,为海产品及水体中致病性溶藻弧菌的检测提供了一种有效方法.     

创伤弧菌、溶藻弧菌外膜蛋白特性的比较研究

对用Sarkosyl法分离的创伤孤菌、溶藻弧菌、副溶血弧菌、鳗弧菌的外膜蛋白进行了初步的比较分析.这4种弧菌外膜蛋白的SDS-PAGE和Western blotting的图谱有相似性亦有差异.SDS-PAGE显示,4种弧菌中除副溶血弧菌外均能分离到47、38 ku的外膜蛋白;创伤弧菌和溶藻弧菌存在4种共同的外膜蛋白,大...     

海南地区溶藻弧菌耐药性及其质粒多样性的研究

调查了海南省的30株溶藻弧菌环境菌株对24种常见抗生素的耐药性,并对菌株携带质粒及其多样性进行了研究,所有菌株对洁霉素、乙酰螺旋霉素、万古霉素、氯洁霉素、制霉菌素5种抗生素完全耐药,对24种抗生素的耐药率为20.8%～66.7%,表明该地区溶藻弧菌具有严重的多重耐药性;30株溶藻弧菌中有22株不携带质粒,8株携带1～3个质粒,质粒为1.20～12.5kb;8株携带质粒的菌株共形成了7种类型的质粒指纹图谱。试验结果表明,该地区溶藻弧菌的耐药性与其是否携带质粒无明显关系。分离菌株的耐药性可能主要是由染色体相关基因引起的。     

斜带石斑鱼溶藻弧菌病的研究

从患病的斜带石斑鱼肝和肾组织分离的两株病原菌,进行纯化培养、人工感染、VITEK-AMS-32自动微生物分析鉴定及药敏试验,经形态和生理生化测定,确定为溶藻弧菌(Vibrioalginolyticus)。组织病理变化主要表现为鳃、肝和肾细胞变性、坏死,病变组织炎性细胞浸润,呈变质性炎症。     

梭子蟹溶藻弧菌病的初步研究

2005～2006年,昌邑池塘养殖梭子蟹发生被称为"牙膏病"的疾病,并导致大量死亡。发病时间主要在7～9月份的高水温期,病蟹典型症状是肌肉白浊、无弹性、呈不透明的乳白色。从病蟹的肌肉、肠道、胃、眼球、肝胰腺均分离到单一形态的微生物,经生理生化鉴定为溶藻弧菌。人工感染实验发现,其对健康梭子蟹具有致病性,出现与自然发病蟹相同的症状,并在人工感染病蟹中分离出同样性状的菌株,证实其为该病病原菌。     

多重PCR在创伤弧菌快速检测中的应用

建立多重PCR方法检测水体及海产品中的创伤弧菌。针对创伤弧菌的gyrB基因的不同位点设计了3对引物,扩增片断的大小分别为481、702、1065bp。该方法的灵敏度为10^2cfu／ml。共对3种海产品进行了创伤弧菌的检测,结果在3种海产品中均能特异性地检测到创伤弧菌。该方法快速,灵敏度高,特异性强。为海产品及水体中的创伤弧菌的检测提供了有效的方法。     

溶藻弧菌减毒活疫苗的构建及其免疫保护性

 采用Overlap PCR和同源重组技术, 结合氯霉素(Cm)正向筛选和sacB基因蔗糖负向筛选, 构建了溶藻弧菌(Vibrio alginolyticus)Ⅲ型分泌系统(Type III secretion system, T3SS)注射装置蛋白vscO基因无标记框内缺失突变株ZJ03ΔvscO。在以石斑鱼(Epinephelus coioides)为模型的感染实验中发现ZJ03ΔvscO的毒力较ZJ03下降约150倍。将ZJ03ΔvscO作为减毒活疫苗通过注射、浸泡的途径免疫石斑鱼(Epinephelus coioides)后, 发现其对溶藻弧菌有很好的保护效应, 其中注射组的免疫保护率达84%, 浸泡组次之(68%)。血清效价分析显示, 免疫后第4周注射组和浸泡组的效价均达到最高值, 分别为1: 2 048和1︰128。上述结果表明, 所构建的ZJ03ΔvscO减毒活疫苗可有效提高石斑鱼对溶藻弧菌的免疫力, 并且免疫和攻毒的途径可能是影响免疫保护效果的因子。本研究旨在为溶藻弧菌引起的鱼类疾病防治提供技术支撑。

     

Application of the rpoS gene for the detection of Vibrio anguillarum in flounder and prawn by polymerase chain reaction

Vibrio anguillarum , an opportunistic fish pathogen, is the main species responsible for vibriosis, a disease that affects feral and farmed fish and shellfish, and causes considerable economic losses in marine aquaculture. In this study, we used polymerase chain reaction (PCR) to detect V. anguillarum . PCR specificity was evaluated by amplifying the rpoS gene, a general stress regulator, in six strains of V. anguillarum and 36 other bacterial species. PCR amplified a species-specific fragment (689 bp) from V. anguillarum . Furthermore, the PCR assay was sensitive enough to detect rpoS expression from 3 pg of genomic DNA , or from six colony-forming units (CFU) mL⁻¹ of cultured V. anguillarum . However, the assay was less sensitive when genomic DNA from the infected flounder and prawn was used (limit of detection, 50 ng and 10 ng g⁻¹ tissue, respectively). These data demonstrate that PCR amplification of the rpoS gene is a sensitive and species-specific method to detect V. anguillarum in practical situations.     

应用聚合酶链式反应(PCR)检测锦鲤疱疹病毒

为建立并优化锦鲤疱疹病毒检测方法,针对目前国内集约化养殖鲤鱼大面积流行的病死现象进行有针对性的检测,对在疫区采集到的病鱼肾脏、肝胰脏、肠等组织,提取基因组DNA,应用锦鲤疱疹病毒特异性引物进行PCR反应,同时设立阴性对照(TE buffer)。通过1%琼脂糖凝胶进行鉴定,与阴性对照比较,在484bp处出现特异性目的条带。经序列测定,所得PCR产物的基因序列与NCBI上锦鲤疱疹病毒的同源率达到99%以上。对同一批次的样品进行重复检测,结果表明该方法具有较好的可重复性,可据此判定结果。通过对锦鲤疱疹病毒的检测,确定了疫区引起框镜鲤(Cyprinus carpio)和建鲤(Cyprinus carpio var Jian)大批死亡的病原为锦鲤疱疹病毒(Koi Herpesvirus,KHV),从而对疫区控制、防治方案的制定提供了理论依据。建议尽快在鲤鱼主要养殖区开展KHV流行病学、诊断与检测及免疫预防等方面的研究。     

Selection and characterization of ssDNA aptamers specifically recognizing pathogenic Vibrio alginolyticus

Vibrio alginolyticus (V. alginolyticus) is a major opportunistic pathogen to both marine animals and humans, which has also caused heavy economic losses to mariculture. The aim of this study was to develop highly specific aptamers for V. alginolyticus. Single‐stranded DNA (ssDNA) aptamers with high binding affinity to viable V. alginolyticus were generated by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and identified by flow cytometric analysis in this study. The selected aptamers showed high specificity for V. alginolyticus and low apparent binding for other bacteria. The aptamers formed distinct stem‐loop structures, which could form the basis of aptamers’ specific binding to the target V. alginolyticus. Aptamer VA2 and VA8 showed particularly high binding affinity constant (Kd) of 14.31 ± 4.26 and 90.00 ± 13.51 nM, respectively. The aptamers produced no cytotoxic effects in vitro and in vivo. ssDNA aptamers were successfully selected against the viable bacteria pathogen V. alginolyticus by SELEX. The aptamers selected in this study could be not only applied as specific chemical molecular probes for studying V. alginolyticus pathogenesis to Trachinotus ovatus, but also developing rapid convenient diagnosis assay for V. alginolyticus infection, even when applied to the complex sample matrix, such as food and environment samples.     

溶藻弧菌相关分离株的分子及VITEK鉴定

哈维群弧菌是弧菌属的核心菌群,包括溶藻弧菌在内的6个种在表型和遗传型上均十分相似,要准确鉴定各种有一定难度。看家基因的研究及生化鉴定系统的出现为弧菌鉴定提供了多种方法,本文比较了16S rRNA基因、toxR基因和pyrH基因以及VITEK 2 COMPACT GN鉴定卡对溶藻弧菌相关分离株的分辨力。哈维群弧菌基因组内16S rRNA基因是多拷贝的,且拷贝间序列差异大于种间差异,不适用于种的鉴定。单拷贝基因toxR和pyrH序列种内差异均小于种间差异。基于toxR基因相似性比较可清楚地将18个疑似溶藻弧菌分离株和5个参比株归并到4个种;toxR基因系统发育学分析也显示,哈维群弧菌各种独立地聚类分支,且溶藻弧菌种内存在两个明显的聚类分支,说明两个分支的溶藻弧菌具有独立的进化方向。pyrH基因的相似性比较和发育学分析也得到类似的结果,但pyrH基因具有较强的保守性,因而分辨力稍低于toxR基因。VITEK 2 COMPACT GN鉴定卡在鉴定溶藻弧菌时也有一定的错误率,且鉴定谱较窄。因此,建议在实际应用中采用toxR基因比对作为溶藻弧菌快速鉴定的主要手段,可再选用pyrH基因对鉴定结果进行验证。     

TaqMan real-time polymerase chain reaction assay for rapid detection of Flavobacterium columnare

Flavobacterium columnare is a ubiquitous Gram-negative bacterium that causes columnaris disease in a wide variety of fish worldwide. Timely detection of this bacterium is important to prevent its spreading and to reduce the economic loss to fish farmers. We developed a TaqMan-based real-time polymerase chain reaction (PCR) targeting a 113 bp nucleotide region of the chondroitin AC lyase gene of F. columnare G₄. Specificity of the assay evaluated with 20 isolates of F. columnare and 15 other taxonomically or ecologically related bacteria revealed that the primers and probe were 100% specific for detection of F. columnare. The sensitivity limit of detection of F. columnare in pure cultures, over a range of dilutions [3.1 × 10⁰–3.1 × 10⁶ colony-forming units (CFU) mL⁻¹], was observed to be ∼3 bacterial cells. The lowest limit of detection in nucleic acids from pure culture of F. columnare was 5.4 fg and the assay was linear with the log of amount of nucleic acid (R²=0.994) over that range (5.4 ng–5.4 fg). In tissues (blood, gills and kidney) of F. columnare experimentally infected fish, the bacterial numbers measured by TaqMan real-time PCR ranged from 3.4 × 10⁰ to 9.5 × 10⁵ CFU mL⁻¹. In both F. columnare experimentally infected and spiked samples, positive PCR results were confirmed by bacteriological culture with 100% agreement. The TaqMan real-time PCR developed in this study is specific, sensitive and reproducible for the detection and quantitation of F. columnare in infected fish.     

三疣梭子蟹“科甬1号”生长速率、形态特征和对溶藻弧菌的耐受性

为探究三疣梭子蟹“科甬1号”新品种(简称“科甬1号”)和普通海捕野生三疣梭子蟹子1代(简称“普通蟹”)在生长速率、形态特征和对溶藻弧菌耐受性上的差异,通过对相同生长条件下 “科甬1号”和普通蟹的30、60、90、120和150日龄5个时间点进行体质量和形态指标测量,并在60、90、120和150日龄4个时间点,用浓度为1.14×10⁷ cfu/mL溶藻弧菌按10 μL/g(体质量)剂量进行体腔注射攻毒并统计死亡率。对数据进行ANOVA检验表明,在全部5个测量时间点,“科甬1号”体质量均极显著大于普通蟹体质量;“科甬1号”和普通蟹各形态特征差异均不显著;在60和90日龄2个攻毒时间点,“科甬1号”溶藻弧菌耐受性均极显著强于普通蟹,在120和150日龄2个攻毒时间点,“科甬1号”溶藻弧菌耐受性均显著强于普通蟹。LSD检验分析表明,在60～150日龄期间“科甬1号”对溶藻弧菌的耐受性较稳定,不随生长时间不同而存在显著差异,此外也表明相同浓度溶藻弧菌对相同品种60～150日龄的三疣梭子蟹根据体质量按10 μL/g剂量进行体腔注射攻毒具有相对稳定的致死率。     

应用多聚酶链反应检测生鸡肉中沙门氏菌的研究

目的:建立快速、特异、灵敏的PCR方法以检测生鸡肉中的肠炎沙门氏菌。方法:以肠炎沙门氏菌的sefA基因作为靶序列设计一对引物,将样品增菌后用煮沸法提取细菌基因组DNA进行检测。结果:每克生鸡肉污染3.0×10°CFU肠炎沙门氏菌,经16h增菌培养后可扩增出500bp特异性性DNA带,对28份屠宰场样品的检测结果与传统方法相符合。结论:PCR法适于生鸡肉中肠炎沙门氏菌的快速、准确检测。     

鳗弧菌O1/O2二价灭活疫苗免疫大菱鲆的抗体持续期和免疫保护期

本研究分析了鳗弧菌(Vibrio anguillarum)O1/O2血清型二价灭活疫苗免疫大菱鲆后的抗体持续期和免疫保护期。以鳗弧菌O1血清型VAM003株和O2血清型VAM007株为抗原制备了福尔马林灭活二价疫苗,将疫苗按照三种剂量(10~7 cells/尾、10~8 cells/尾、10~9 cells/尾)以腹腔注射途径免疫大菱鲆,在免疫后3 d、7 d、14 d、30 d、60 d、90 d、120 d、150 d,用血清凝集实验检测了免疫鱼血清的VAM003和VAM007抗体效价,用攻毒实验检测了疫苗的免疫保护率(RPS)。结果显示,在免疫后7 d三个剂量组的大菱鲆均产生了特异抗体,并获得27%～60%的RPS。三个剂量组大菱鲆的O1血清型抗体持续期分别90 d (10~7 cells/尾组)、150 d (10~8 cells/尾组)、150 d (10~9cells/尾组),而三个剂量组大菱鲆的O2血清型抗体持续期均150 d。三个剂量组的大菱鲆获得的免疫保护持续期均150 d;以RPS75%为有效免疫保护,各剂量组大菱鲆抵抗O1血清型病原感染的有效免疫保护期为:14～120d(10~7 cells组)、14～120 d (10~8 cells/尾)、14～150 d (10~9 cells/尾),抵抗O2血清型病原感染的有效免疫保护期为:14～60 d (10~7 cells组)、14～120 d (10~8 cells/尾)、14～120 d (10~9 cells/尾)。研究结果表明鳗弧菌二价灭活疫苗可为大菱鲆提供有效而稳定的免疫保护,获得的抗体持续期和免疫保护期为该疫苗的临床中试研究提供了基础。     

Seasonal dynamics in a coastal Vibrio community examinedby a rapid clustering method based on 16S rDNA

ABSTRACT:   A rapid clustering scheme for Vibrio specieswas developed based on the analysis of 16S rDNA, which compriseda group-specific polymerase chain reaction (PCR) and restriction fragmentlength polymorphisms (RFLP) of the PCR-amplified 16S rDNA. The group-specificPCR, using a specific primer Vib1F, clustered the Vibrio speciesinto two large groups: Vib1F⁺ group and Vib1F^– group.The PCR-RFLP, using Sca I and Bln I, further dividedthese groups into smaller groups. Finally, 46 Vibrio speciesand eight related species could be clustered into 14 groups usingthis rapid clustering method. Seasonal dynamics of the Vibrio communityin Yoshimi Bay, Hibiki-nada Sea, Japan, were examined based on therapid clustering method. In both seawater and sediments, the groupcomprised Vibrio parahaemolyticus , Vibrio alginolyticus , Vibriocampbellii , Vibrio carchariae , Vibrio harveyi and Vibrionatriegens and was predominant when the seawater temperaturewas above 20°C, whereas the group of Vibrio splendidus biotypeI and Vibrio lentus was abundant when the temperature wasaround or below 20°C.     

Detection of Koi herpesvirus: impact of extraction method,primer set and DNA polymerase on the sensitivity of polymerase chain reaction examinations

Since virus isolation is seldom successful, KHV infection is commonly detected by PCR examination. A number of different PCR assays have been described in recent years. However, at present no commonly accepted PCR method is used amongst different laboratories. The aim of this study was to check if the examination of infected fish by different PCR methods yielded comparable results. We used tissue samples of three KHV‐infected koi, one KHV‐infected common carp, one KHV‐infected goldfish and one non‐infected common carp. DNA was extracted with DNAzol Reagent, High Pure PCR Template DNA Preparation Kit and QIAamp DNA Mini Kit. The DNA was tested by PCR with different combinations of published primer sets –KHV‐F and ‐R, KHV‐Gray‐2F and ‐2R and KHV‐TKf and ‐TKr – plus different DNA polymerases – a standard Taq DNA polymerase, a Platinum (hotstart) Taq DNA polymerase and a Platinum (hotstart) Pfx DNA polymerase with proofreading activity. The different extraction methods produced DNA solutions with different yields of DNA and different degrees of homogeneity. Also, the sensitivity of the PCR depended on the choice of the primer set and polymerase. Not all infected fish could be identified with all methods; there were large differences in the sensitivity between methods.