同时检测海产品中副溶血弧菌和霍乱弧菌的合检方法评价研究

目的建立水产品中副溶血弧菌和霍乱弧菌合检方法,并对合检方法对比进行评价。方法利用副溶血弧菌和霍乱弧菌阳性菌株,制备纯菌液、不同浓度梯度的人工污染样品。通过对30份纯菌液、30份人工污染样品和250份实际样品的检测,将建立的合检方法与行业标准(SN/T 1022-2010进出口食品中霍乱弧菌检验方法和SN/T 0173-2010进出口食品中副溶血性弧菌检验方法)进行比较,对合检方法进行效果评价。结果实验结果表明副溶血弧菌和霍乱弧菌合检方法,与行业标准检测结果完全一致。结论该方法可靠,对实验仪器和操作人员的要求低,具有良好的实用性,适合基础检测实验室。     

超高压处理对牡蛎中副溶血性弧菌的消减作用研究

为探索有效的消减副溶血性弧菌（Vp）的超高压处理条件,利用CDC-1（临床分离株,血清型O3:K6）和CGMCC1.1997（标准菌株,血清型O1:K1）两种菌株,研究了不同Vp经超高压处理的消减变化情况;比较了CDC-1菌株接种到牡蛎体内与混合到牡蛎肉中的超高压处理的差异性。结果显示,超过200MPa的压力对两种Vp均有明显的消减作用,增大压力和延长处理时间可以提高超高压处理对Vp的消减作用;多重比较显示两种Vp对超高压的耐受力存在显著差异（p<0.05）,CDC-1较CGMCC1.1997有更强的超高压耐受力;超高压处理对两种接种方式Vp的消减效果存在显著差异（p<0.05）,接种到牡蛎体内的CDC-1菌株的耐受力高于混合在牡蛎肉中的CDC-1菌株;用300MPa处理10min或350MPa处理5min能有效的消减牡蛎中的Vp。      

LAMP技术快速检测海产品副溶血弧菌的条件优化

本研究以副溶血弧菌不耐热溶血素tlh为目标基因设计特异性引物,优化反应条件,并对特异性、灵敏度进行了验证。LAMP最佳反应条件为,外内引物浓度比为1∶8,Mg2+反应浓度为4 mmol/L,d NTPs浓度为3.5 mmol/L,温度为65℃,反应时长为1 h,只对副溶血弧菌产生扩增反应,与其余细菌不产生扩增反应,细菌基因组DNA和纯培养物的灵敏度约为5.45×101 fg/μL和140 cfu/m L,对人工污染食品样品进行检测,检出限为2.8×102 cfu/m L。该方法反应时间短、特异性强、灵敏度高。      

北京口岸进口鲜活海产品中副溶血性弧菌致病性分析

目的调查从北京口岸进口的鲜活海产品中是否存在致病性副溶血性弧菌。方法对2005年从北京口岸进口的鲜活海产品中分离的267株副溶血性弧菌（Vibrio parahaemolyticus）的尿素酶活性、神奈川现象和毒性基因（tdh和trh）进行了检测。结果267株副溶血性弧菌中27株尿素酶呈阳性,其中14株菌tdh基因和砌基因呈阳性。tdh基因和trh基因呈阳性的14株菌中有10株菌神奈川现象为阳性,并且全部分离自从加拿大进口的象拔蚌。结论北京口岸进口的鲜活海产品中存在致病性副溶血性弧菌,主要集中在象拔蚌中。应对从加拿大进口的象拔蚌加强监管,防止致病性副溶血性弧菌引起食物中毒发生。     

养殖海水贝类中副溶血性弧菌的致病性及 耐药性分析

目的对从山东和辽宁沿海地区养殖海水贝类中分离到的84株副溶血性弧菌进行致病性及耐药性分析。方法通过PCR扩增及测序法检测毒力基因,通过神奈川试验测定溶血能力,采用K-B法进行药敏试验分析。结果 84株菌均含有tlh基因,均不含trh基因,有1株菌含tdh基因。tdh基因阳性菌株的神奈川试验呈阳性,其余菌株均呈阴性。菌株对氨苄西林、头孢呋辛钠和复方新诺明的耐药率分别为91.7%、6.0%和1.2%。所有菌株对阿莫西林/克拉维酸、头孢曲松、头孢吡肟、阿米卡星、庆大霉素、亚胺培南、氧氟沙星、环丙沙星、萘啶酸、氯霉素、氟苯尼考和呋喃妥因高度敏感。结论海水贝类中含有少量致病性副溶血性弧菌,菌株存在一定程度的耐药性,提示应加强对海产品中副溶血性弧菌致病性及耐药性的监控。     

大连市不同海产品中副溶血性弧菌污染的健康风险分级研究

目的对大连市不同海产品中副溶血性弧菌(Vibrio parahaemolyticus, VP)污染的健康风险进行分级和评价。方法 2017年1～10月,在大连市10个县市区分层随机采集4类944份常见海产品。依据GB 4789.7—2013《食品安全国家标准食品微生物学检验副溶血性弧菌检验》进行海产品中VP定量检测,使用实时荧光定量聚合酶链式反应(PCR)方法进行VP毒力基因检测;采用食物频率法获得各类海产品的消费量数据;利用专家咨询法获得海产品交叉污染和烹调习惯参数;采用快速微生物定量风险评估(sQMRA)方法,对4类海产品中致病性VP的健康风险进行分级。结果甲壳类导致人体感染致病性VP发病风险和年发病例数最高,分别为3.5×10~(-6)和2 799.3例。鱼类导致人体感染致病性VP发病风险和年发病例数居第二位,分别为1.1×10~(-6)和1 304.4例。海产品导致人群VP发病的主要途径为交叉污染。结论应关注大连市甲壳类中VP对人群的致病风险,重点控制海产品在加工处理过程中VP的交叉污染。     

Incidence and Enumeration of Vibrio parahaemolyticus in Shellfish from Two Retail Sources and the Genetic Diversity of Isolates as Determined by RAPD-PCR Analysis

A total of 83 shellfish samples from two local retail sources (A and B) yielded 38 samples positive for the presence of Vibrio parahaemolyticus based on 3 tube MPN enrichments and isolation from thiosulfate-citrate-bile salts-sucrose agar (TCBS) and biochemical tests. The 38 positive samples yielded 133 biochemically presumptive isolates of V. parahaemolyticus. Among these 133 presumptive isolates, 104 were confirmed by the polymerase chain reaction (PCR), which yielded more reliable identification results than the biochemical tests. The 38 biochemically presumptive samples yielded 29 samples that were confirmed by PCR to be positive for the presence of V. parahaemolyticus. RAPD analysis with three random primers was performed to examine the genetic diversity of 64 strains among the PCR confirmed V. parahaemolyticus isolates from both retail sources. 52 of 56 composite RAPD types consisted of single strains, indicating that most of the V. parahaemolyticus isolates were genetically quite heterogeneous. No strains representing the same RAPD type occurred in both retail outlets, implying that contamination of the shellfish by V. parahaemolyticus from the 2 retail sources was from different environmental locals and shellfish harvesting areas. Eight genomic clusters were generated at the 25% similarity level in a dendrogram based on RAPD profiles. With few exception, isolates with close genetic relationships grouping into an individual cluster tended to be derived from the same retail source.     

双层平板直接定性定量法检测海产品中副溶血性弧菌

目的:建立一种直接定量定性检测海产品中副溶血性弧菌的方法。方法:参照国标菌落计数方法,筛选具备计数和显示副溶血性弧菌典型菌落颜色功能的培养基,将人工染菌样品分别置于50、4和-18℃温度下并分别进行测试。结果:在NaCl NA双层平板上副溶血性弧菌菌落呈淡紫色,在人工染菌样品实验中与NaCl NA平板计数无显著差异;NaCl NA双层平板直接定量检测4℃冷藏处理和-18℃冷冻的样品中副溶血性弧菌浓度分别是对照组的68.9%和21.7%。结论:NaCl NA双层平板法具备操作简便、检验周期短、结果稳定、灵敏度高等特点,适合应用于-18~50℃温度环境下放置的海产品中副溶血性弧菌直接定性定量检测,能如实反映样品受污染的情况。     

Incidence and Enumeration of Vibrio parahaemolyticus in Shellfish from Two Retail Sources and the Genetic Diversity of Isolates as Determined by RAPD-PCR Analysis

A total of 83 shellfish samples from two local retail sources (A and B) yielded 38 samples positive for the presence of Vibrio parahaemolyticus based on 3 tube MPN enrichments and isolation from thiosulfate-citrate-bile salts-sucrose agar (TCBS) and biochemical tests. The 38 positive samples yielded 133 biochemically presumptive isolates of V. parahaemolyticus. Among these 133 presumptive isolates, 104 were confirmed by the polymerase chain reaction (PCR), which yielded more reliable identification results than the biochemical tests. The 38 biochemically presumptive samples yielded 29 samples that were confirmed by PCR to be positive for the presence of V. parahaemolyticus. RAPD analysis with three random primers was performed to examine the genetic diversity of 64 strains among the PCR confirmed V. parahaemolyticus isolates from both retail sources. 52 of 56 composite RAPD types consisted of single strains, indicating that most of the V. parahaemolyticus isolates were genetically quite heterogeneous. No strains representing the same RAPD type occurred in both retail outlets, implying that contamination of the shellfish by V. parahaemolyticus from the 2 retail sources was from different environmental locals and shellfish harvesting areas. Eight genomic clusters were generated at the 25% similarity level in a dendrogram based on RAPD profiles. With few exception, isolates with close genetic relationships grouping into an individual cluster tended to be derived from the same retail source.     

双层平板法对海产食品中副溶血性弧菌定量效果评价

评价了双层平板(DLAP)计数法对海产食品中副溶血性弧菌的定量分析效果。通过在混合菌悬液和鱼、虾、贝等生物体中人为模拟染菌,并进行冷冻受伤减菌处理,采用DLAP计数法对副溶血性弧菌处理前后的菌量变化进行分析,同时采用MPN法、BCVM平板计数法和TCBS平板计数法3种方法作为对照,运用SPSS软件分析4种方法测定结果的显著性差异(P<0.05)。试验结果表明,在理想的控制条件下,4种方法对不同基质条件中副溶血性弧菌的测定结果不存在明显差异;在菌体活力不强或受伤状态下,DLAP计数法对该菌的测定结果与MPN法在同一水平上且缩短了5d的检验周期,其准确性优于BCVM平板计数法和TCBS平板计数法,具有省时、准确的特点,适合广普应用。     

食品中副溶血性弧菌的危害分析、检测与预防控制

副溶血性弧菌是一种广泛分布于近岸海水、海底沉积物和海产品中的嗜盐性细菌，也是引起我国特别是沿海地区细菌性食物中毒危害的首要食源性致病菌。为了提高人们对副溶血性弧菌的重视程度，降低食源性疾病的风险，本文就副溶血性弧菌的生物特性、毒力因子、污染途径、食物中毒特点、检测方法以及预防措施作简要综述。     

高产AI-2副溶血性弧菌的筛选及毒力特性研究

副溶血性弧菌是一种常见的食源性致病菌,其运动能力、生物被膜形成和毒力因子产生等受AI-2/LuxS型群体感应系统调控。本研究从9株副溶血性弧菌中筛选具有高产AI-2信号分子能力的菌株,通过添加外源AI-2信号分子分析其对生物被膜、毒力因子和运动性等的影响。结果表明,获得1株高产AI-2信号分子的副溶血性弧菌VP-4,添加不同体积分数的外源AI-2信号分子(1%~7%)对其生物被膜、明胶酶、卵磷脂酶、淀粉酶、运动能力和溶血性的促进率分别为33.02%~67.87%,1.67%~7.26%,3.25%~48.15%,1.21%~12.17%,4.92%~35.98%,4.15%~50.60%,2.06%~15.23%。同时,扫描电镜观察发现添加外源AI-2信号分子的副溶血性弧菌生物被膜菌体聚集堆积,厚度增加,结构致密。上述结果说明AI-2信号分子可以促进副溶血性弧菌生物被膜的形成及毒力因子的释放,提高其运动能力。本研究可为控制海产品中副溶血性弧菌的致病性提供新靶点。     

风险分级矩阵在贝类海产品中副溶血性弧菌风险评估的应用研究

目的 研究风险分级矩阵方法学应用以及我国沿海地区居民贝类海产品中副溶血性弧菌污染的健康风险等级.方法 利用贝类海产品消费量以及副溶血性弧菌污染等数据,计算危害严重性(5分制)和疾病发生可能性(5分制)参数,导入风险分级模型矩阵,对我国沿海地区不同人群副溶血性弧菌健康风险进行赋值和等级评价.结果 我国沿海地区全人群和贝类...     

Vibrio parahaemolyticus in granulated ark shell clam (Tegillarca granosas): accumulation from water and survival during cold storage and thermal process

This study investigated accumulation of Vibrio parahaemolyticus in granulated ark shell clam (Tegillarca granosas) exposed to contaminated water and survival of V. parahaemolyticus in the clams during cold storage and heating processes. Vibrio parahaemolyticus could be accumulated in clams to a level similar to that of contaminated water within 12 h of exposure of clams to contaminated water at temperatures between 9 and 33 °C. Keeping clams stored at 5 and 0 °C for 10 days resulted in 1.98 and 2.32 log MPN g^?1 reductions of V. parahaemolyticus, respectively, in the clams. Frozen storage at ?18 °C for 15 days or at ?30 °C for 30 days were capable of reducing V. parahaemolyticus from 4.05 log MPN g^?1 to non‐detectable levels (< 3 MPN g^?1). A heating process in hot water at 80 °C or higher for 1 min also reduced V. parahaemolyticus in the clams to non‐detectable levels.