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.     

1株副溶血性弧菌Bh-06的分离、鉴定及其药敏试验

从患病的南美白对虾分离出1株副溶血性弧菌Bh-06,通过革兰染色和Biolog全自动细菌鉴定仪鉴定,并对健康南美白对虾进行攻毒试验和对该菌株进行药物敏感试验。试验结果表明:Bh-06菌株为革兰阴性弧菌,通过细菌自动鉴定仪鉴定为副溶血性弧菌;该菌在培养第5小时进入对数早期,对数期一直延续到25小时;该菌在1.0×106cfu/mL浓度时可引起南美白对虾发病,而且浓度越高,病症越严重;该菌对氧哌嗪青霉素产生高度的敏感性。     

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%。     

浙江沿海地区海产品及环境中副溶血弧菌的分离与主要毒力基因分析

2007~2008年间, 我们调查了浙江沿海地区海产品和养殖环境中副溶血弧菌的污染状况, 并分析了不同来源副溶血弧菌中主要毒力相关基因tdh、trh、ureC和T3SS2(vscC2、vcrD2)的分布特征及溶血表型与尿素酶表型。结果显示, 566份样品中共分离到395株副溶血弧菌, 检出率高达70%, 毒力相关基因分析结果发现, tdh基因阳性率为10.1%, trh与ureC基因阳性率分别为 20.0%与 11.1%, 40株tdh+菌中组成T3SS2的vscC2基因阳性率为32.5%, 其中38株tdh+菌的神奈川试验亦呈阳性; 但在44株trh+-ureC+菌株中, 尿素酶表型阳性只有6株。试验表明, 浙江沿海地区海产品及其养殖环境中副溶血弧菌污染状况比较严重, 且有相当比例的菌株携带毒力或疑似毒力基因。研究结果为深入探索副溶血弧菌的致病性、基因结构与功能(或表型)及其分子演化提供基础。     

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...     

实时荧光PCR检测水产品中副溶血性弧菌

目的探索副溶血性弧菌快速检测法,应用于日常监测及食物中毒的快速查源。方法用副溶血性弧菌实时荧光试剂盒对水产品样本进行检验,以副溶血性弧菌toxR基因为靶序列,设计1对引物和探针,采用热裂解法提取DNA。结果实时荧光PCR从42份水产品样品的增菌液中检出13份样品副溶血性弧菌阳性,与传统培养法相比一致性极好（K=0.943,K〉0.75）。结论实时荧光PCR方法在副溶血性弧菌的检验方面较传统方法具有快速、灵敏、特异性强等优势,具有广阔的应用前景。     

生食贝类水产品中副溶血性弧菌的半定量风险评估

为研究上海市生食贝类水产品的食用安全性,进行了半定量风险评估。牡蛎、醉泥螺、象拔蚌、北极贝为上海市主要的生食贝类水产品,调查显示贝类水产品中副溶血性弧菌平均检出率为43.7%,其中副溶血性弧菌致病性基因Tdh+/Trh+在贝类水产品中的平均检出率为3.75%。对上海地区对生食贝类的消费情况进行了膳食调查,并利用"Risk Ranger"软件对牡蛎、醉泥螺、象拔蚌、北极贝中副溶血性弧菌进行了半定量风险评估。上海市生食贝类消费人群比例为13.86%,生食贝类的食用频率平均2.4月/餐次,平均每餐消费生食贝类水产品为49.06 g/餐。风险评估结果显示:牡蛎/副溶血性弧菌(50)属于高度风险,醉泥螺/副溶血性弧菌(46)、象拔蚌/副溶血性弧菌(44)、北极贝/副溶血性弧菌(39)均属于中度风险,根据风险排序风险大小依次为牡蛎醉泥螺象拔蚌北极贝。     

酒、盐度及酸碱度对醉泥螺中副溶血性弧菌生长的影响

醉泥螺中的副溶血性弧菌对人有较高的致病风险,为了提高醉泥螺的食用安全性,本文对其腌制工艺进行了优化,探讨了酒、盐度和酸碱度三个因素对醉泥螺中副溶血性弧菌生长状况的影响,并用描述性检验对盐度不同的醉泥螺进行了感官评价。研究结果表明,52%vol白酒,6. 6%盐度,酸碱度p H 5. 5的工艺条件能够最高效地降低醉泥螺腌制过程中副溶血性弧菌引起的食用安全风险,副溶血性弧菌的生长最受抑制。但兼顾不同盐度醉泥螺的感官评价,本研究认为52%vol白酒,4. 4%盐度,酸碱度pH 5. 5是最适合腌制醉泥螺的加工条件。     

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分离与计数

副溶血性弧菌(副弧菌)是世界沿海国家的重要食物中毒菌,因而也是食品卫生细菌学检验的主要项目之一。海产品与副弧菌食物中毒密切相关。为了适应进出口商品检验的需要,我们以海产品进行了副弧菌检验方法的实验探索,现将结果报告如下:     

海洋蛭弧菌的分离鉴定及其对副溶血弧菌的作用

蛭弧菌广泛存在于自然水体, 具有噬菌的特性, 对水体中细菌数量控制具调节作用。以副溶血弧菌为宿主菌, 利用双层琼脂法, 从海洋水体中分离出15株具有噬菌作用的细菌, 对形成噬菌斑能力最强的1株菌株进行特异性16S rDNA扩增, 确认为蛭弧菌, 命名为Bd-M1。Bd-M1对大多数海水养殖动物病原菌有裂解作用, 裂解率在90%(20/22)以上, 模拟水环境实验发现, 蛭弧菌对副溶血弧菌有较强的裂解和净化作用, 102 h内能使副溶血弧菌从3.0′108 CFU/mL下降到8.7×103 CFU/mL。动物实验表明蛭弧菌能有效预防对虾弧菌病的发生, 表明蛭弧菌有望成为水产动物疾病防治的一种有效的生物制剂。     

Isolation and partial characterization of a compound with siderophore activity from Vibrio parahaemolyticus

A compound with siderophore activity was purified by successive column and thin layer chromatographic procedures from Dowex 1 x 8 extracts of culture supernatants of Vibrio parahaemolyticus AQ 3354. The strain synthesized the compound in culture media containing less than 2 microM added FeCl3. Hydrolysis of the compound yielded alanine, ethanolamine, citric acid and 2-ketoglutaric acid. The 1H-NMR spectrum exhibited the presence of a residue from each of these components in the intact molecule. The fast-atom bombardment mass spectrum of the methyl ester derivative indicated a prominent ion at m/z 477, probably corresponding to [M + 1] ion. Other strains of V. parahaemolyticus were also found to produce this compound when grown in an iron-limited medium.     

副溶血弧菌CHY5-M1M噬菌体的分离鉴定及生物学特性分析

副溶血弧菌引起的疾病给水产养殖行业带来巨大损失,而随着抗生素的禁用,人们开始探索治疗水产动物病菌的新型方法,如噬菌体治疗法。从青岛市城阳水产品批发市场采集了15份海产品养殖水及下水道污水样品,以副溶血弧菌作为宿主菌,采用点滴法分离纯化获得12株副溶血弧菌噬菌体,通过双层平板法研究副溶血弧菌噬菌体CHY5-M1M的最佳感染复数、一步生长曲线、温度和pH的稳定性以及对紫外线的敏感度等生物学特性。结果显示,CHY5-M1M噬菌体效价为8.65×10¹³CFU·mL^-1,且在100感染复数时效价最高;一步生长曲线的潜伏期为20 min,裂解时长100 min,140min后达到平稳期;噬菌体在温度为40℃时效价最高,高于60℃时活性开始下降;噬菌体pH适应范围为5～11;噬菌体浓度随紫外照射时间增加明显下降;噬菌体基因组共43 193 bp,包含44个基因,无毒力因子基因和抗生素耐药基因。研究结果表明,噬菌体CHY5-M1M在开发新型抗弧菌药物、预防治疗严重海水动物弧菌病等方面具有良好的应用前景,有望作为未来的抗生素替代产品。     

一株副溶血弧菌噬菌体的分离鉴定及生理特性

为了探寻有效的控制副溶血弧菌的方法, 从水产品市场的污水中分离出来一株烈性噬菌体qdvp001。采用双层平板法分离纯化噬菌体qdvp001, 电镜观察其形态特征, 并利用双层平板法测定其生理特性, 包括热稳定性试验、最适pH、最佳感染复数及一步生长曲线, 然后提取基因组进行酶切和序列分析。结果显示该烈性噬菌体qdvp001头部直径大约为79 nm, 尾长大约118 nm, 属于肌尾噬菌体科。它对60 °C以下的温度耐受力较强, 最适pH为7.0?8.0左右, 最佳感染复数是0.000 1, 感染宿主菌的潜伏期约20 min, 裂解期约70 min, 并获得部分DNA片段的序列。将获得的DNA序列在NCBI上进行比对, 结果显示, qdvp001与其他噬菌体的同源性较低。该噬菌体很可能是一种新发现的噬菌体。     

A novel multiplex PCR for the identification of Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus

AIM: To establish a simple multiplex polymerase chain reaction (PCR) that will identify Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. METHODS AND RESULTS: A total of 429 Vibrio spp. from various origins were tested with the novel primers targeting toxR. The reverse primers were all designed to be species specific, while the forward primer was universal. The primers correctly identified all the V. parahaemolyticus, V. cholerae and V. vulnificus isolates tested. CONCLUSIONS: The toxR multiplex PCR works well when the initial colony morphology is known. If not, Vibrio alginolyticus might represent a diagnostic obstacle. SIGNIFICANCE AND IMPACT OF THE STUDY: The method provides a fast and reliable way of identifying the main Vibrio spp. involved in food-borne disease. The method could prove very useful for laboratories working with identification of these Vibrio spp.     

Comparison of Vibrio parahaemolyticus hemolysin (Vp-TRH) produced by environmental and clinical isolates

TDH-related hemolysin (Vp-TRH) produced by Kanagawa-phenomenon-negative (KP-) Vibrio parahaemolyticus has been demonstrated to be a possible virulence determinant. Though almost half of KP- isolates examined from diarrhoeal patients produced Vp-TRH, few reports mentioned the ability of environmental isolates to produce Vp-TRH. Considering the route of infection with V. parahaemolyticus, this toxin must be produced by the organisms in the sea or in sea food. To confirm that Vp-TRH produced by V. parahaemolyticus could be involved in sea-food-borne diarrhoeas, Vp-TRH-producing strains were isolated from the environment, identified and hemolysin purified from these strains was compared to hemolysin (Vp-TRH) isolated from diarrhoeal patients. The results showed that the hemolytic activity, antigenicity, reactivity in the rabbit ileal loop test and N-terminal amino acid sequence of Vp-TRH from environmental strains was indistinguishable from the toxin of clinical origin.     

Identification of tdh-positive Vibrio parahaemolyticus from an outbreak associated with raw oyster consumption in Spain

Between August and September 1999, a total of 64 cases of illness were identified in three episodes of acute gastroenteritis associated with the consumption of live oysters from a typical outdoor street market in Galicia (northwest Spain). Nine case patients were hospitalized and analysis of their stool samples revealed the presence of Vibrio parahaemolyticus. The strains isolated from two stool samples were studied for antibiotic susceptibility, biochemical characteristics and presence of virulence factors. Both isolates were Kanagawa phenomenon positive and produced thermostable direct hemolysin, which is related to pathogenicity in humans. These results show the presence of pathogenic V. parahaemolyticus in mollusks harvested in Europe and reveal the risk of illness associated with their consumption, suggesting the revision of V. parahaemolyticus risk assessment associated with consumption of raw live shellfish.     

四种副溶血弧菌总RNA提取方法的比较

副溶血弧菌(Vibrio parahaemolyticus)是一种革兰氏阴性嗜盐菌.提取高质量的RNA是研究副溶血弧毒力基因表达与其致病性和环境适应性的基础.本研究分别用SDS法、Trizol法,PureLinkTM RNA Mini Kit和Uniq-10柱式Trizol总RNA抽提试剂盒提取六株副溶血弧菌的总RNA,用普通琼脂糖凝胶电泳和核酸测定仪检测RNA的质量,并用RT-PCR法对四种提取方法进行比较.研究结果表明,Trizol法和PureLinkTM RNA Mini Kit简单快捷,提取的总RNA完整性好,纯度高,可用于后续实验的分子生物学研究;而Trizol法更适用于普通实验室细菌RNA的提取.