副溶血性弧菌显色培养基检测效果初步评价

副溶血性弧菌(Vibrio parahaemolyticus)是一种重要的食源性致病菌,广泛存在于各种海产品中.由于传统培养基和检测方法费时费力,本研究设计开发了一种新型显色培养基HKC vibrio,通过应用于人工污染样品和实际样品检验,以法国科玛嘉弧菌显色平板CHROMagar vibrio和柠檬酸钠-硫代硫酸钠-氯化钠-蔗糖琼脂平板(TCBS)为对照,对显色培养基HKC vibrio的灵敏性、特异性和检测效果进行了初步评价.结果表明,HKC vibrio的灵敏性与CHROMagar vibrio和TCBS相当,并具有较好的特异性,HKC vibrio是非常有价值的分离平板,可大大提高副溶血性弧菌的检测效率.     

一种副溶血性弧菌显色培养基的应用

副溶血性弧菌(Vibri oparahemolyticus)是一种在海产品中广泛存在的嗜盐性细菌,也是重要的食源性致病菌。传统检验方法检测周期长、特异性低,为提高检测效率、缩短检测周期,开发出一种副溶血性弧菌显色培养基(HKMVPM),与国外同类产品KHVPM和TCBS在灵敏度、特异性及检测效果方面进行了比较和初步评价。结果显示,HKMVPM显色培养基和KHVPM显色培养基均具有较好的选择性和特异性,这3种培养基对天然污染样品的分离率高低顺序为HKMVPMKHVPMTCBS。显色培养基具有高检出率及高特异性的特点,有良好的应用前景。     

两种检测水产品中副溶血性弧菌方法的比较分析

为了比较传统检测方法与快速检测方法的优劣,本实验室采用国标法、显色培养基+API20E法对50组水产品进行副溶血性弧菌的检测,结果两种方法检测结果一致,即样品S006、S043中检出副溶血性弧菌,检出率为4%,其余样品未检出该菌。显色培养基+API20E法在检测时间和操作步骤方面要优于传统检测方法。     

海产品中副溶血性弧菌的直接定量法-疏水网格滤膜法

本研究建立了一种疏水网格滤膜法,直接定量检测海产品中的副溶血性弧菌。该方法包括4~5 h复苏步骤以使受损细胞得以修复,然后37℃下在副溶血性弧菌显色培养基上培养过夜后计数。从显色培养基上挑取典型菌落进行确证试验,确证率大于98%。与传统MPN法比较,对经过冷藏、冷冻和热处理的样品进行检测时,新方法检出菌量明显高于MPN法(p0.01)。     

辛辛那提弧菌选择性鉴别培养基的研制

旨在设计开发一种更适用于海产品重要致病菌——辛辛那提弧菌快速筛选与检测的选择性鉴别培养基(VciDM)。基于辛辛那提弧菌特有的综合酶系统及其代谢特点和对某些抑菌剂的抵抗力,设计开发出一种VciDM,用于辛辛那提弧菌的反复检验,对VciDM的显色效果、平板效率、灵敏度和特异性进行评价。VciDM对辛辛那提弧菌具有较好的选择性和特异性;在37°C±0.1°C培养16-24h,辛辛那提弧菌菌落为黄色;其他致病菌菌落呈绿色或不生长;VciDM对辛辛那提弧菌的出菌率高,为(82.14±4.45)%;VciDM的检出限为101CFU/mL;与常规方法相比,结果易于观察,减少了溶藻弧菌等常伴弧菌的干扰,省去了TCBS分离培养后的生化试验,降低检测成本,减少工作量,具有更高的检测效率和鉴别能力。VciDM平板可以对辛辛那提弧菌进行快速监测。     

副溶血性弧菌在青岛近海及几种海产食品中的微生态分布

已知副溶血性弧菌(Vibrio parahaemolyticus)广泛分布于海洋环境中,它也是一种水产食品的重要食物中毒菌及海产动物的条件性病原菌(Opportunistic pathogen)。本文报告了在青岛近岸海水中和几种常见的水产食品污染了该菌情况,并在检测方法上,修定了一种选择性较强的培养基(SAC),实验结果表明“SAC”较其他常用的几种培养基对副溶血性弧菌检出的阳性率和灵敏性均较高,与常用的“TCBC”法比较,其     

致病性副溶血性弧菌菌落杂交检测体系的优化

对常见食源性致病菌副溶血性弧菌的杂交条件进行了优化。基于副溶血性弧菌的tdh,trh,toxR基因选用了3种探针序列,从菌落杂交样品的预处理方法、杂交时间和显色检测等方面对基于副溶血性弧菌毒力基因的原位杂交实验进行了条件优化。结果表明,采用80℃热固定2 h,37℃预杂交10 min后杂交8 h以及封闭1 h的改良方法可获得高特异性、低背景且着色清晰的实验结果。优化的菌落杂交技术检测副溶血性弧菌与传统检测方法相比,具有高效、准确、可区分致病性菌株的优点,为水产品中副溶血性弧菌致病菌株和非致病菌株的同步检测和筛选提供参考。     

四重PCR检测副溶血性弧菌及其毒力基因方法的建立

【目的】建立同时检测副溶血性弧菌tox R、tdh、trh、tlh基因的四重PCR快速检测方法。【方法】分别以副溶血性弧菌的tox R、tdh、trh、tlh 4个基因为靶基因,设计4对特异性引物,对4对引物浓度和退火温度进行优化,获得最佳引物比例和扩增条件,建立快速检测致病性副溶血性弧菌的四重PCR体系。通过特异性验证、灵敏度验证以及模拟样品检测进行方法确认。【结果】四重PCR体系扩增条带与预期相符,即115 bp(tox R)、244 bp(tdh)、418 bp(trh)、759 bp(tlh)4个目的条带;用74株副溶血性弧菌和37株非目标菌的测试结果表明,所建立的方法有良好的特异性。该方法对模板DNA的检测灵敏度为50μg/L,纯培养物的检测灵敏度为6.7×103 CFU/m L;副溶血性弧菌含量为1.36 CFU/g的人工模拟样品增菌6 h后,tox R、tlh、tdh、trh 4个基因可同时被检出。【结论】该方法可实现同时检测携带tox R、tdh、trh、tlh 4种基因的副溶血性弧菌,对开展致病性副溶血性弧菌的检测研究具有一定现实意义。     

海产品中副溶血性弧菌的分离、鉴定及与临床分离株的生化性状比较

利用TCBS和科玛嘉两种选择性培养基从60份海产品中初筛得到27株疑似副溶血性弧菌, 经生化鉴定, 27株均为副溶血性弧菌, 其中22株是从鲜活海产品中分离得到, 5株从冷冻海产品中分离得到。进一步与临床上得到的32株副溶血性弧菌进行了四个特殊生化试验的比较, 结果发现副溶血性弧菌临床分离株与海产品分离株在溶血、脲酶及枸橼酸盐利用3个生化试验上存在差异, 而阿拉伯糖利用试验无差异, 这为根据生化性状研究不同来源副溶血性弧菌提供基础。     

海产品中副溶血性弧菌的分离、鉴定及与临床分离株的生化性状比较

利用TCBS和科玛嘉两种选择性培养基从60份海产品中初筛得到27株疑似副溶血性弧菌,经生化鉴定,27株均为副溶血性弧菌,其中22株是从鲜活海产品中分离得到,5株从冷冻海产品中分离得到.进一步与临床上得到的32株副溶血性弧菌进行了四个特殊生化试验的比较,结果发现副溶血性弧茵临床分离株与海产品分离株在溶血、脲酶及枸橼酸盐利用3个生化试验上存在差异,而阿拉伯糖利用试验无差异,这为根据生化性状研究不同来源副溶血性弧菌提供基础.     

Comparative evaluation of a chromogenic agar medium-PCR protocol with a conventional method for isolation of Vibrio parahaemolyticus strains from environmental and clinical samples

Screening for pathogenic Vibrio parahaemolyticus has become routine in certain areas associated with food-borne outbreaks. This study is an evaluation of the CHROMagar Vibrio (CV) medium-PCR protocol and the conventional method (TCBS (thiosulfate-citrate-bile salts-sucrose) agar plus biochemical and Wagatsuma agar tests) for detection of V. parahaemolyticus in shrimp, water, sediment, and stool samples collected for biosurveillance in an endemic area of northwestern Mexico. A total of 131 environmental and clinical samples were evaluated. The CV medium-PCR protocol showed a significantly improved ability (P < 0.05) to isolate and detect V. parahaemolyticus, identifying isolates of this bacteria missed by the conventional method. Although some other bacteria, distinct from pathogenic V. parahaemolyticus, produced violet colonies similar to that of V. parahaemolyticus on CV medium, we were able to detect a superior number of samples of V. parahaemolyticus with the CV medium-PCR protocol than with the conventional method. The Kanagawa phenomenon is routinely determined on Wagatsuma agar for the diagnosis of V. parahaemolyticus (pathogenic) positive for thermostable direct hemolysin (TDH) in developing countries. In our results, Wagatsuma agar showed low sensitivity (65.4% at 24 h and 75.6% at 48 h) and specificity (52.4% at 48 h) for identifying V. parahaemolyticus positive for TDH. Overall, our data support the use of the CV medium-PCR protocol in place of the conventional method (TCBS-biochemical tests-Wagatsuma agar) for detection of pathogenic V. parahaemolyticus, both in terms of effectiveness and cost efficiency.     

A comparison of thiosulphate-citrate-bile salts-sucrose (TCBS) agar and thiosulphate-chloride-iodide (TCI) agar for the isolation of Vibrio species from estuarine environments

AIMS: The purpose of this study was to compare a recently described medium, thiosulphate-chloride-iodide (TCI), for the isolation of estuarine vibrios with thiosulphate-citrate-bile salts-sucrose (TCBS). METHODS: A total of 492 colonies which developed on these media from estuarine water samples taken monthly over a 10-month period were examined. RESULTS: A much larger number of colonies developed on TCBS than TCI, and minimal taxonomic criteria indicated that a higher percentage (61%) of TCBS colonies could be identified as Vibrio spp. when compared with TCI (46%). SIGNIFICANCE: This study suggests that TCBS is a superior medium when compared with TCI for the isolation of Vibrio spp. from estuarine waters. Because of the public health risk presented by V. vulnificus, V. parahaemolyticus, V. cholerae and other vibrios, the selection of the most appropriate medium for their isolation is extremely important.     

海水及海产品中溶藻弧菌的分离与鉴定

从上海东海海域随机采集海水样品50份及各市场购买海产品95份,参照国标中副溶血性弧菌的检验方法,对采集样品进行了分离与鉴定。样品增菌后选用TCBS及科玛嘉弧菌显色培养对其进行初步分离,挑取可疑单菌落进行生理生化验证和分子生物学方法鉴定,其中从9份海水、24份海产品中分离出溶藻弧菌菌株,分离率分别为18.0%与25.3%。     

青岛近岸海水及几种海产品中副溶血弧菌(Vibrio parahaemolyticus)生境的调查

副溶血弧菌是水产食品中的一种重要食物中毒菌,它在沿岸海水和海产动物体上都有广泛的分布,所以它又是海产动物的一种条件性致病菌。本文报告了该菌在青岛近岸海水和几种常见的水产食品上污染了该菌的情况,在检测方法上也进一步修定了一种选择性较强的培养基(SAC)。调查发现青岛近岸海水中副溶血弧菌的出现率为68～463CFU/100ml,几种常见海产食品中平均含量值为745～6900CFU/100克,其中贝类含量最高达6900/100克。采用常用的选择培养基“TCBS”等与修定的“SAC”对该菌进行检测比较,结果表明“SAC”法较“TCBS”法检出的阳性率和灵敏性均较高,两种方法X~2=4.28,P>0.05,效果有显著的意义。     

Growth of heat-injured Vibrio parahaemolyticus in media supplemented with various cations.

Mid- to late logarithmic growth phase cells of Vibrio parahaemolyticus grown in tryptic soy broth (TSB) containing 0.5, 3.0, and 7.5% NaCl were heated for 8 min at 45 degrees C in 0.1 M phosphate buffer (pH 7.2) containing 3% NaCl. Colony formation on thiosulfate-citrate-bile salts-sucrose agar (TCBS) containing 2% NaCl was greatest for unheated cells that had been grown in 7.5% NaCl-TSB; cells grown in 0.5% NaCl-TSB formed a greater number of colonies on 1.0% NaCl-TCBS. Thermal injury was evident in heated cells, regardless of the NaCl concentration in TSB growth medium. The effects of Mg2+, K+, and Li+ added as chlorides to 0.5% NaCl-TSB on the growth of nonheated and heated V. parahaemolyticus were studied. Lower levels of Mg2+ and slightly higher levels of K+ were required to replace Na+ in TSB inoculated with thermally injured cells that had been originally grown in 3.0 and 7.5% NaCl-TSB. LiCl had an inhibitory effect on both nonheated and heated cells when present in the recovery medium (0.5% NaCl-TSB) at concentrations as low as 0.5%. Increased numbers of colonies were formed by heated cells plated in MgCl2-supplemented TCBS, regardless of the NaCl concentration in the original growth medium. Potassium had little, if any, effect on colony formation by nonheated V. parahaemolyticus recovered on TCBS and may have had a detrimental effect on heat-injured cells.     

基于组合胶体金纳米颗粒的免疫层析试纸条快速可视化检测海产品中副溶血性弧菌

本研究以野生型副溶血性弧菌(Vibrio parahaemolyticus)为免疫原制备单克隆抗体,并以组合胶体金纳米粒子(CP-AuNPs)为抗体标记探针,开发了一种基于双抗体夹心原理的可视化快速检测副溶血性弧菌的免疫层析试纸条方法.该方法被应用于梅子鱼、鲜虾、白蛤等海产品中副溶血性弧菌的检测,具有特异性强、检测时间...     

副溶血弧菌LAMP检测方法的建立

副溶血弧菌(Vibrio parahaemolyticus)是一种能引起食源性疾病的重要病原菌。首次将一种新颖的核酸扩增技术-环介导等温扩增技术（Loop-Mediated Isothermal Amplification, LAMP）应用于副溶血弧菌的快速检测。针对副溶血弧菌不耐热溶血毒素基因（tlh）设计四条特异性引物（两条内引物和两条外引物）进行LAMP扩增,对扩增反应进行优化,最佳反应时间为60 min,反应温度为60 ℃。对12种细菌共28株菌进行LAMP扩增,仅14株副溶血弧菌得到阳性扩增结果,证明引物具有很高的特异性。副溶血弧菌基因组DNA和纯培养物的检测灵敏度分别约为90 fg和24 cfu/mL。对模拟食品样品进行直接检测,检测限为89 cfu/g。结果表明,该方法检测副溶血弧菌特异性强、灵敏度高,并且操作简便、检测成本低,1 h即可完成,有望发展成为快速检测副溶血弧菌的有效手段。     

Sensitivity of the vibrios to ultraviolet-radiation.

The ultraviolet-inactivation kinetics of a number of strains of Vibrio cholerae (classical), Vibrio cholerae (el tor), NAG vibrios and Vibrio parahaemolyticus were investigated. Statistical analyses revealed significant differences between any two of the four types of vibrio in respect to their sensitivity to U.V.