用PCR检测副溶血性弧菌耐热溶血基因

根据副溶血性弧菌的耐热血素基因设计了２对引物、经聚合酶链式反应检测，所有１８株神奈川试验阳性副溶血血性弧菌均呈阳性反应，而６株神奈川试验阴性副溶血性弧菌呈阴性反应，其中９种弧菌和８株其他革兰氏阴性菌也呈阴性反应。     

实时荧光PCR快速检测水产品中副溶血性弧菌的研究

目的建立快速检测水产品中副溶血性弧茵的实时荧光PCR方法。方法利用副溶血性孤菌TDH基因的保守序列设计引物和探针,建立快速检测副溶血性弧菌的实时荧光PCR方法。结果本研究建立的检测副溶血性弧菌的实时荧光PCR方法,其纯茵检测低限低于10cfu/PCR反应体系;对模拟阳性样品直接检测,检测低限为10~3cfu/g。模拟样品经增茵培养4～6h后,检测低限达1cfu/g;用本法对24株标准菌株/参考菌株进行检测,结果除副溶血性弧菌呈阳性外,其他23株非副溶血性弧菌均呈阴性反应;重复性试验结果:批内和批间变异系数均小于1%。结论本研究建立的副溶血性弧菌实时荧光PCR方法具有快速、灵敏、特异和重复性好的优点,可在8h内对水产品中的副溶血性弧菌进行定性或定量检测。     

副溶血弧菌SHJLA株的分离鉴定及致病性分析

副溶血性弧菌（Vibrio parahaemolyticus）是海洋环境中常见的食源性致病茵。本研究从对虾中分离出1株细菌SHJLA,在TCBS和弧菌显色培养基上分别显示典型的蓝绿色和紫红色的菌落,且其生理生化特性具有典型副溶血弧菌的特性。以SHJLA菌株的基因组为模板。检测副溶血弧菌种特异性基因（tlh、toxR、groEL）均为阳性,gyrB基因序列分析表明SHJLA与副溶血弧茵的亲缘关系最近,同源性达98％～100％;检测副溶血弧菌主要毒力相关基因tdh和T3SS2（VopC2和vcrD2）均为阳性。该菌的神奈川溶血实验为阳性,对小鼠的半数致死量（LD50）为4．8×10^8 cfu／mL。结合SHJLA菌株的形态、生理生化、种特异性基因的检测、gyrB基因序列分析、毒力相关基因的检测以及小鼠半数致死量的测定结果,确定SHJLA是一株携带毒力基因的副溶血弧菌。     

市售仔虾中副溶血性弧菌的检测

应用常规细菌分离法对西宁市某几个农贸市场采集的70份仔虾样品进行了副溶血性弧菌的检测,经细菌形态观察、嗜盐性试验、细胞色素氧化酶试验和生化特性测定,结果鉴定为副溶血性弧菌。检出阳性菌株4株,阳性率为5.7%（4/70）,同时通过致病性试验证明4株分离菌对小白鼠有致死效应。     

依赖于核酸序列恒温扩增技术快速检测副溶血性弧菌方法的建立

为建立副溶血性弧菌的快速检测方法,本研究采用依赖于核酸序列恒温扩增(NASBA)技术,以副溶血性弧菌的tlh基因为扩增的靶基因设计特异性引物和探针,建立可快速扩增副溶血性弧菌的NASBA方法.特异性和灵敏度试验结果表明:该NASBA方法对副溶血性弧菌的最小检出量为5.1×102 cfu/mL,高于普通PCR方法,而且与其他种属的菌无任何交叉反应.此外,本研究将副溶血弧菌扩增产物采用通用型核酸扩增物快速检测板进行检测,实现了特异性强的快速副溶血弧菌的检测.该方法对仪器要求低,具有良好的应用前景.     

舟山沿海贝类产品中副溶血性弧菌的检测与毒力基因分析

为了解舟山沿海贝类产品中副溶血性弧菌污染情况,我们于2008年3个不同季节,从不同的农贸市场采集贝类标本,采用实时荧光定量PCR检测方法和常规培养方法,对舟山沿海贝类产品中的副溶血性弧菌进行了检测,同时对分离到的菌株进行血清学分型和毒力基因检测。结果60份贝类产品中,采用常规的分离培养方法,阳性率为83.33%,而采用实时荧光定量PCR检测方法,全部检出副溶血性弧菌。对分离到的50株副溶血性弧菌进行耐热溶血素(tdh)基因检测,结果4株tdh阳性。监测结果表明舟山贝类海产品中携带副溶血性弧菌情况比较严重,应引起足够的重视。     

8株方斑东风螺病原菌对17种中草药敏感性测定

采用抗菌中药体外药敏试验的平皿打孔法,测定了17种中草药对2株副溶血性弧菌、1株哈氏弧菌、1株创伤弧菌、1株腐败希瓦氏菌、1株鲍鱼希瓦氏菌、2株海藻希瓦氏菌共8株菌的抑菌作用;选取其中对供试菌株具有抑菌作用的11种中草药,进行两两联合药敏试验,以研究其抗菌相互作用关系。结果显示,五倍子、乌梅、苏木、猴耳环4种中草药对8株供试菌株呈高度敏感;大黄、地榆、桉叶3种中草药对8株菌呈中度敏感;黄芩、石榴皮、连翘、秦皮4种中草药除分别对一株菌腐败希瓦氏菌、哈氏弧菌、创伤弧菌、副溶血性弧菌1呈低度敏感外,对其余7株菌均呈中度敏感;石韦、鸭胆子、益智仁、郁金、板蓝根、贯众6种中草药除石韦对一株海藻希瓦氏菌呈中度敏感外,其余均呈低度敏感。联合药敏试验中除五倍子与桉叶、黄芩、秦皮,乌梅与黄芩、地榆,苏木与大黄、石榴皮,桉叶与黄芩,大黄与黄芩,地榆与秦皮等对药对8株菌中的某些菌株呈协同作用外,其余均呈相加作用,未见无关和拮抗作用。     

副溶血性弧菌PCR检测方法的建立

为了建立适于基层单位应用的针对副溶血性弧菌检测的特异PCR方法,检测副溶血性弧菌的基因序列.针对该菌的属特异性基因t1基因进行引物设计,扩增片段大小为449 bp.运用该PCR法可特异性的从副溶血性弧菌ATCC17802标准株中扩增出目的基因片段,与GenBank上发表的序列同源性为100%.而经常污染海产品的溶藻弧菌,嗜水气单胞茵标准株J-1,铜绿假单胞茵标准株ATCC27853结果均为阴性.该PCR法最低检出菌体DNA量为10-2ng以及最低检出菌数为5.7 × 103 CFU/mL.对临床上送检的35份样品进行菌体DNA PCR方法检测并同时与国标GB/T4789.7-2003中使用的检测方法进行比对,两种检测方法的结果完全符合,与国标中使用的检测方法相比可大大缩短鉴定时间.因此该PCR法能快速鉴定当前副溶血性弧菌流行群,有利于流行病学溯源等研究.     

广东省候鸟副溶血性弧菌的分离与鉴定

为了解广东省候鸟副溶血性弧菌及其携带的毒力基因、抗生素敏感性和遗传进化特征,收集广东省候鸟粪便,用国标法和toxR基因PCR检测的方法进行分离鉴定获得副溶血性弧菌;通过PCR对毒力基因进行鉴定、用Etset测定MIC值,通过ERIC-PCR对副溶血性弧菌进行遗传进化分析。结果共获得122株副溶血性弧菌,这些细菌仅含有tlh(不耐热溶血素基因),而tdh(耐热溶血素基因)与trh(耐热溶血素相关毒素基因)等均为阴性。通过ERIC分群发现福田红树林保护区与湛江的鹭源菌株和湛江雷州的鹭源菌株具有高度相似性,深圳华侨城湿地菌株分布在3群,与雷州的鹭源相似度较高,为95%;福田红树林保护区分离株与深圳华侨城湿地菌株相似度大于75%。广东省候鸟携带的副溶血性弧菌具有较为丰富的遗传多样性,毒力基因携带单一,保持着较高的药物敏感性。     

苏州大型超市小水产品中副溶血性弧菌的污染调查及耐药性分析

目的了解副溶血性弧菌(Vibrio parahaemolyticus)在苏州市大型超市小水产品中的污染状况及耐药性,为建立副溶血性弧菌食物中毒预警系统提供科学依据。方法定期从苏州一大型超市抽取小水产品,根据GB/T4789.7-2008标准进行副溶血性弧菌的分离培养和鉴定,应用K-B法进行药敏实验。结果在144份样品中检出副溶血性弧菌56株,检出率38.9%。所有分离菌株对先锋必素、诺氟沙星和环丙沙星敏感;部分菌株对氨苄西林、阿莫西林、复方新诺明和头孢噻吩等具有较强的耐药性,耐药率分别为55.4%、42.9%、35.7%和32.1%;有9株菌株出现了多重耐药。结论苏州大型超市小水产品中副溶血性弧菌污染严重,应加强副溶血性弧菌食源性疾病预警,同时加强水产品抗生素使用的管理,防止其耐药菌株的传播。     

基于toxR基因的SYBR GreenⅠ实时定量PCR检测病原副溶血弧菌方法的建立

基于副溶血弧菌toxR基因保守序列设计1对特异性引物,建立了SYBR GreenⅠ实时定量PCR检测副溶血弧菌的方法。常规PCR检测,副溶血弧菌扩增出大小为368bp的目的片段,其他4种病原细菌均为阴性;SYBRGreenⅠ实时定量PCR扩增曲线反映了PCR的指数增长阶段和平台阶段;在Tm为85℃,扩增产物的熔解曲线分析只出现1个单特异峰,无引物二聚体;所制作的标准曲线在2.7×108～2.7×102拷贝数之间有较好的线性关系,相关系数为0.998,能对副溶血弧菌进行准确的定量分析;该方法检测时间从核酸抽提到结果分析仅需4～5h,较传统方法敏感、操作简单,耗时短,是副溶血弧菌引起的水产动物疾病的快速诊断及食品中针对副溶血弧菌安全检测的有效手段。     

荧光定量PCR法检测副溶血弧菌tdh基因的表达差异

以pvuA为内标基因,运用实时荧光定量PCR检测不同来源以及不同应激条件下副溶血弧菌热稳定直接溶血素基因tdh的表达量。pvuA和tdh基因的荧光定量PCR融解曲线分析表明,两者均为特异性扩增。尽管相同来源的不同菌株间tdh表达量存在显著差异,副溶血弧菌临床分离株的tdh mRNA平均表达量显著高于海产品分离株((57.2比13.8)。在pH4.0、0.5%和8%NaCl应激条件下,临床株ZJ2和海产品分离株FJ14A的tdh mRNA表达量显著高于对照组;另一海产品分离株KP34在8%NaCl条件下的表达量显著提高,而低pH应激时tdh mRNA的表达量显著降低。结果表明,不同副溶血弧菌分离株的tdh mRNA表达差异显著,临床分离株的tdh mRNA表达量总体上高于海产品分离株,副溶血弧菌在不同应激条件下主要表现为tdh mRNA表达上调。     

Detection of Vibrio parahaemolyticus in Mediterranean mussels (Mytilus galloprovincialis) in Slovenia

The aim of this study was to determine the prevalence of Vibrio parahaemolyticus in shellfish samples harvested along the Slovenian coast. Shellfish samples of Mediterranean mussels (Mytilus galloprovincialis) were collected along the Slovenian coast at four locations (Se?a, Piran, Strunjan and Debeli Rti?) between 2006 and 2008. Samples were examined and analysed for the presence of V. parahaemolyticus by conventional and molecular methods. The presence of Vibrio in the samples was examined by conventional methods on plate grown bacterial cells before and after enrichment in alkaline saline peptone water (ASPW). PCR methods were used for the detection of V. parahaemolyticus-specific toxR and tlh genes and of the virulence-associated tdh and trh genes. Out of 168 samples examined, 24 were positive for toxR and tlh genes by PCR from enrichment broth. Five out of 62 (8.1%), 4 out of 32 (12.5%) and 15 out of 74 (20.2%) samples were positive in 2006, 2007 and 2008, respectively. Colonies of V. parahaemolyticus were isolated from only one sample positive for V. parahaemolyticus by PCR.     

Attachment of Vibrio parahaemolyticus strains to estuarine algae.

Attachment of Vibrio parahaemolyticus strains to estuarine microalgae was examined in artificial seawater by viable counts of the organism and direct counts of the bacterial cells after immunoperoxidase staining. Thermostable direct hemolysin (TDH)-producing and TDH-non-producing strains of V. parahaemolyticus were found to attach to five estuarine strains of Navicula (diatom alga) in similar levels. The level of the bacterial attachment depended on salinity and temperature of the water, in which the maximum attachment was observed in 15% artificial seawater at 25 degrees C, a typical condition of Hashizu estuary in Japan during summer months. The attachment was inhibited by pectinase digestion of the algal cells. These evidences confirmed the participation of the microalgae to the ecological cycle of V. parahaemolyticus at the estuary.     

Plasma-dependent chemotactic activity of hemocytes derived from a juvenile estuarine gastropod mollusc, Clithon retropictus, to Vibrio parahaemolyticus and Escherichia coli strains.

Hemocytes of adult Clithon retropictus were attracted chemotactically to live Vibrio parahaemolyticus and Escherichia coli strains. The chemotaxis was stimulated by the plasma of adult C. retropictus. Hemocytes of the juvenile specimen were attracted chemotactically to V. parahaemolyticus and E. coli strains in the presence of the plasma of the juvenile and only to E. coli strain in the absence of the plasma. These evidences suggest that hemocytes of juvenile C. retropictus might be defective to recognize V. parahaemolyticus strains and that the hemocytes would display full activities in the presence of the plasma factor(s).     

Isolation of Vibrio vulnificus and atypical Vibrio from surface water of the Baltic Sea in Germany

From 1995 to 1997 several defined species like V. alginolyticus, V. anguillarum, V. cholerae (non O1 and non O139), V. mimicus, V. parahaemolyticus and V. vulnificus were isolated during a survey to determine the presence of V. vulnificus in the brackish water of the Baltic Sea in Germany. Moreover atypical Vibrio isolates were detected. Four isolates belonging to a group of atypical Vibrio and possibly representing a new species in the genus Vibrio were characterized in detail. All four strains were isolated from surface costal waters. Based on 16S rDNA sequence analysis they showed the highest relatedness to the species V. navarrensis and V. vulnificus. The strains did not harbor the species specific hemolysin gene vvhA from V. vulnificus as shown by PCR and hybridization experiments. Moreover, they differed in at least two biochemical parameters tested from the hitherto described Vibrio species. All these strains induced hemolysis on washed blood agar dishes and showed phase variations on Luria Bertani agar dishes. Because of the similarity to the eel pathogen V. vulnificus, we infected eels with one of the four atypical strains (CH-291), but no pathogenicity for eels could be detected. Furthermore, Vero cell tests with supernatants of bacterial cultures did not reveal secreted Vero cell cytotoxic compounds. This indicates a nonpathogenic nature of these strains.     

北海近岸养殖海域3种弧菌的分离鉴定及耐药性分析

对北海近岸养殖海域的3种弧菌(Vibrio)进行了分离鉴定,并对鉴定的弧菌进行耐药性分析。通过从北海近岸养殖海域随机采集水样,利用TCBS培养基对所采水样进行弧菌的分离和纯化,采用PCR方法和序列分析对弧菌进行鉴定,并对鉴定出的3种弧菌进行了常用抗菌药物耐药性的检测。共分离获得67株疑似弧菌菌株,经鉴定,19株为副溶血性弧菌(Vibrio parahaemolyticus),4株为霍乱弧菌(Vibrio cholera),4株为河流弧菌(Vibrio fluvialis)。耐药性分析研究表明,19株副溶血性弧菌总体显示对10种常用抗菌药物具有耐药性,其中100%的菌株对四环素、卡那霉素和红霉素表现耐药,94.7%的菌株对氨苄西林耐药、84.1%的菌株对左氧氟沙星和78.9%的菌株对环丙沙星耐药;4株霍乱弧菌和4株河流弧菌对10种抗菌药物的耐药性均较强,但敏感度低,两者对复方新诺明均表现为中介敏感。本研究提示,北海近岸养殖海域中弧菌种类较多,其中以副溶血性弧菌为主,大多对常用抗菌药物具有耐药性,对北海近岸海水养殖疾病防治具有一定的指导意义。     

Chemotactic activity of hemocytes derived from two marine neritid gastropod molluscs, Nerita albicilla and Heminerita japonica, to Vibrio parahaemolyticus and Escherichia coli strains.

Hemocytes of two marine neritid gastropods, Nerita albicilla and Heminerita japonica, were attracted chemotactically to live Vibrio parahaemolyticus and Escherichia coli strains. Chemotactic attraction of N. albicilla hemocytes was enhanced in the presence of N. albicilla plasma, while that of H. japonica hemocytes was not enhanced in the presence of H. japonica plasma. Chemotactic activity of the hemocytes seems to participate in the rapid elimination of V. parahaemolyticus from these gastropods.