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。     

湛江两近海域文蛤和翡翠贻贝副溶血性弧菌污染情况分析

目的:对湛江东海岛和南三岛两近海域文蛤和翡翠贻贝副溶血性弧菌的污染进行检测和评价。方法:在5～11月,采用SN0173-92出口食品副溶血性弧菌检验方法,分别对文蛤和翡翠贻贝样品进行副溶血性弧菌生化反应初筛和MPN值测定。结果:每克文蛤样品中副溶血性弧菌的MPN值为<3～93,其中在6～7月副溶血性弧菌MPN值最高,为93。每克翡翠贻贝样品中副溶血性弧菌的MPN值<3～23,其中在6～7月副溶血性弧菌MPN值最高,为23。结论:湛江东海岛和南三岛附近海域每克文蛤和翡翠贻贝样品中副溶血性弧菌MPN最高值≤100,未超过相关国际标准。     

上海市零售梭子蟹中副溶血性弧菌的污染状况及风险评估

目的 研究上海市场零售梭子蟹中副溶血性弧菌的污染状况,并对副溶血性弧菌可能引发的公共卫生风险进行初步评估.方法 采用最可能数(MPN)法检测上海和宁波市售梭子蟹中副溶血性弧菌的带菌量,采用膳食回顾性调查方法对上海居民梭子蟹消费量进行调查,采用Risk Ranger软件对梭子蟹中副溶血性孤菌进行丰定量风险评估.结果 上海和宁波市零售梭子蟹中副溶血性弧菌阳性率分别为60.5%和13.3%,阳性试样平均菌量分别为0.75 MPN/g和5.50 MPN/g.膳食调查结果显示,上海居民梭子蟹人均日摄入量为2.95 g/d.风险评估结果显示,风险评分为36分,梭子蟹食用者每人每天的发病概率为8.22×10-9,每年上海地区预期发生42.5例副溶血性弧菌感染病例.通过改变评估模型中某些风险的选择,如采取良好的控制措施,风险可降低至原来的1/10.结论 上海市售梭子蟹副溶血性弧菌污染状况较为严重,可能对人群健康造成较高的风险,未来有必要进一步加强监测.     

2008—2010年上海市夏秋季市售海产品中副溶血性弧菌污染监测结果分析

目的 了解上海市夏秋季节市售海产品中副溶血性弧菌的污染水平和特征.方法 2008-2010年5-10月,采用GB/T 4789.7-2008《食品卫生微生物学检验副溶血性孤菌检验》方法,对上海市批发市场、集贸市场、卖场超市和餐饮单位等污染物监测点的市售海产品进行副溶血性弧菌的定性和定量检测.结果 共监测市售海产品941件,副溶血性弧菌总体检出率为13.2％,不同种类、不同监测月份和不同采样地点的海产品,其副溶血性弧菌检出率和样品几何平均浓度总体上差异有统计学意义(P＜0.05).其中,海产虾类副溶血性弧菌检出率(25.0％)和样品几何平均浓度(5.0 MPN/g)显著高于其他类海产品(P＜0.05);8月份海产品副溶血性弧菌检出率(27.4％)和样品几何平均浓度(3.3 MPN/g)显著高于其他监测月份(P＜0.05);集贸市场副溶血性弧菌检出率(28.5％)和批发市场样品几何平均浓度(3.9 MPN/g)显著高于其他采样地点(P＜0.05).结论 上海市市售海产品中副溶血性孤菌的污聚率较高,应进一步开展海产品中副溶血性弧菌的风险监测和评估,并针对副溶血性弧菌污染的高风险环节开展监管.     

茶多酚对泥蚶中副溶血性弧菌的抑制效果

为了降低食用泥蚶引起的副溶血性弧菌食源性疾病的风险,研究了天然抑菌剂茶多酚对泥蚶中副溶血性弧菌的抑制作用并做出感官评价。副溶血性弧菌的定量采用三管最大可能值法(MPN法)。研究结果表明,茶多酚对副溶血性弧菌的最低抑制浓度(MIC)为0.5 g/L;带菌泥蚶经0.5 g/L的茶多酚处理后于0℃,5℃和10℃下贮藏60h,副溶血性弧菌数量分别下降了2.75 Log10 MPN/g,2.59 Log10 MPN/g和2.15 Log10 MPN/g,杀菌率均在99%以上。经感官评定小组评定,茶多酚浓度为0.5 g/L时,对泥蚶的色泽、气味、口感等感官特性不产生负面影响。茶多酚是一种理想的天然抑菌剂。     

生食动物性水产品中副溶血性弧菌和创伤弧菌污染状况分析

目的 了解生食动物性水产品中副溶血性弧菌和创伤弧菌污染状况。方法 采用随机抽样方法,在我国13个地区的餐饮店、零售店和批发市场采集生食动物性水产品,共计2 980份,对样品进行副溶血性弧菌和创伤弧菌检测。结果 生食动物性水产品中副溶血性弧菌检出率为14.7%(437/2 980),污染水平＞100 MPN/g的样品比例为2.9%(83/2 909);创伤弧菌检出率为3.5%(104/2 980)。采样于批发市场的样品中副溶血性弧菌检出率、污染水平＞100 MPN/g的样品比例和创伤弧菌检出率均高于餐饮店和零售店。第三季度副溶血性弧菌检出率、污染水平＞100 MPN/g的样品比例和创伤弧菌检出率最高。造成污染的主要原因包括原产地污染,储存不当及加工过程交叉污染。结论 生食动物性水产品中存在副溶血性弧菌和创伤弧菌的污染,其健康风险应引起关注,本次污染状况分析可为标准制修订提供理论依据。     

改良方法检验冷冻海产品中副溶血性弧菌

【目的】建立一种有效、可靠的方法检验冷冻海产品中副溶血性弧菌，弥补传统方法的局限。【方法】用改进碱性胨水(MAPW)和盐结晶紫增菌液(SVPE)同时对5种冷冻海产品50份样进行增菌后，分别接种至科玛嘉弧菌显色培养基(CV)和硫代硫酸钠柠檬酸胆盐蔗糖培养基(TCBS)，以评估两种增菌液和两种分离培养基的效果；随机选取改良方法检出的10个分离株，用PCR技术扩增副溶血性弧菌特有的t1基因，以认证改良方法的准确性。【结果】当采用改良方法，即MAPW增菌CV分离时，检出率为100％；当采用传统法即SVPE增菌TCBS分离时，检出率为60～80％；用PCR技术在所选取的10个分离株中都扩增出了t1基因。【结论】改良方法是一种高效、准确的检验方法。     

水产品中副溶血性弧菌计数的不确定度评定

目的建立水产品中副溶血性弧菌MPN法计数结果的不确定度评定方法。方法依据JJF1059.1-2012测量不确定度评定与表示和SN/T4091–2015食品微生物学测量不确定度评估指南,分析检测过程中不确定度主要来源,计算合成不确定度和扩展不确定度。结果显示样品制备、递增稀释和加样过程引起的不确定度所占分量较小,属于B类不确定度,重复性试验引起的不确定度属A类不确定度。计算得出95%的置信水平的扩展不确定度为0.082,检验结果为81 MPN/g时,取值范围为(81±7)MPN/g。结论本文采用的评定方法,可以对水产品副溶血性弧菌MPN法计数结果的不确定度做出估计,用于水产品副溶血性弧菌污染的安全性评估。     

副溶血性弧菌噬菌体的分离及其在即食虾中的应用

为了研究噬菌体作为天然生物防控剂应用于食品中的前景,本实验从水产品市场的污水中采用双层平板法分离纯化出一株烈性副溶血性弧菌噬菌体SHOU24,测定其生理特性,并探讨其在即食虾中的抑菌效果。结果表明:该噬菌体只对含有毒力基因tdh的副溶血性弧菌有裂解能力。电镜观察其形态表明该噬菌体属于长尾噬菌体科。SHOU24的最适p H为7~8。在50℃温度以下,存活率很高。抑菌实验结果表明该噬菌体在常温条件下对即食虾中副溶血性弧菌的生长具有良好的抑制作用,能使副溶血性弧菌的菌浓度下降1log CFU/g。本研究为副溶血性弧菌噬菌体SHOU24作为抑菌剂应用于食品中提供了一定的理论基础。      

多重荧光定量PCR法检测海产品中副溶血性弧菌、沙门菌和单增李斯特菌

目的建立检测海产品中副溶血性弧菌、沙门菌和单增李斯特菌的多重荧光定量PCR体系。方法针对副溶血性弧菌tlh基因,沙门菌Ompc基因和单增李斯特菌hly基因设计引物和Taq Man探针,建立多重荧光定量PCR体系,进行特异性与敏感性研究;利用该体系检测海产品中的副溶血性弧菌、沙门菌和单增李斯特菌。结果副溶血性弧菌、沙门菌和单增李斯特菌可得到特异性扩增,而共存于海产品中的其他细菌均未见扩增曲线。敏感性试验显示,该体系对副溶血性弧菌、沙门菌和单增李斯特菌的最低检测限分别为72、40、80 cfu/ml。对舟山采集的150份样品进行检测,检出32份副溶血性弧菌、11份沙门菌、5份单增李斯特菌,与国标法检测结果一致。结论本研究建立的基于Taq Man探针的多重荧光定量PCR检测方法可以特异、灵敏、简单快速地实现对海产品中副溶血性弧菌、沙门菌和单增李斯特菌的检测。     

巢式PCR快速检测海产品中的副溶血弧菌

副溶血弧菌是一种世界范围性的食源性致病菌,食用了该菌污染的海产品可导致胃肠炎等疾病。为了建立一种可快速、特异地检测海产品中副溶血弧菌的方法,通过把副溶血弧菌基因组序列和其它不同种类弧菌的基因组序列进行比较分析,筛选出了一个副溶血弧菌特异性的标记基因-VP1331,根据该基因建立了副溶血弧菌的巢式PCR快速检测方法,并评估了其特异性、敏感性和稳定性。实验结果表明,该方法只有在以副溶血弧菌基因组DNA为模板时才能扩增出目的片段,而其它11种弧菌和非弧菌均不能扩增出目的片段。该方法的最低检测限为副溶血弧菌基因组DNA 10 fg、纯培养物6.6 CFU。人工污染实验表明,初始菌液浓度为25.7 CFU/100 mL时只需经过2 h的增菌培养即可检出。上述结果表明,VP1331基因可以作为副溶血弧菌种特异性标记,本方法可以用于污染海产品中该菌的检测与鉴定。     

Development and evaluation of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Vibrio parahaemolyticus

Loop-mediated isothermal amplification (LAMP) assays targeting the rpoD and toxR genes were developed to detect Vibrio parahaemolyticus. All 78 tested V. parahaemolyticus strains yielded positive results within 40 min, while negative results were obtained for 69 strains of other organisms even at 60 min. For V. parahaemolyticus ATCC 17802 in pure culture, the detection limits of LAMP assays targeting rpoD and toxR were 3.7 and 450 CFU per test, respectively. Due to the higher sensitivity of rpoD-LAMP, it was further evaluated for the ability to detect V. parahaemolyticus in seafood samples. V. parahaemolyticus populations spiked in short-necked clams were enumerated by the most-probable-number (MPN) method combined with the rpoD-LAMP assay and the MPN method with a culture method using agar medium. The MPN-rpoD-LAMP method had better sensitivity and was more rapid than the conventional method. These results indicate that the MPN-LAMP assay targeting the rpoD gene is a specific, sensitive, and rapid method to enumerate V. parahaemolyticus organisms.     

Increased sensitivity in PCR detection of tdh-positive Vibrio parahaemolyticus in seafood with purified template DNA

PCR is an important method for the detection of thermostable direct hemolysin gene (tdh)-positive (pathogenic hemolysin-producing) strains of Vibrio parahaemolyticus in seafood because tdh-negative (nonpathogenic) V. parahaemolyticus strains often contaminate seafood and interfere with the direct isolation of tdh-positive V. parahaemolyticus. In this study, the use of PCR to detect the tdh gene of V. parahaemolyticus in various seafoods artificially contaminated with tdh-positive V. parahaemolyticus was examined. PCR was inhibited by substances in oysters, squid, mackerel, and yellowtail but not by cod, sea bream, scallop, short-necked clam, and shrimp. To improve detection, DNA was purified by either the silica membrane method, the glass fiber method, or the magnetic separation method, and the purified DNA was used as the PCR primer template. For all samples, the use of the silica membrane method and the glass fiber method increased detection sensitivity. The results of this study demonstrate that the use of properly purified template DNA for PCR markedly increases the effectiveness of the method in detecting pathogenic tdh-positive V. parahaemolyticus in contaminated seafood.     

Development of a quantitative real-time PCR method for estimation of the total number of Vibrio parahaemolyticus in contaminated shellfish and seawater

A real-time PCR method targeting the toxR gene of Vibrio parahaemolyticus was developed to quantify the number of V. parahaemolyticus cells, including those of both the hemolysin-producing and nonproducing strains. The specificity of the primer and probe set was confirmed using 25 strains of V. parahaemolyticus and 30 strains of other microbial species. We determined the threshold cycle number using the real-time PCR and the number of V. parahaemolyticus cells by plate count using serially diluted pure culture and developed a standard curve for quantification. Standard curves for V. parahaemolyticus in seawater and seafood were established using artificially inoculated samples. The threshold cycle number and the number of V. parahaemolyticus cells were correlated with 10(1) to 10(7) CFU/ml in pure culture, seawater, and shellfish homogenate. The real-time PCR method developed in this study was compared with the most-probable-number method in seafood samples that were naturally contaminated. The differences in the number of V. parahaemolyticus cells as determined by the culture method and the PCR method were less than 10-fold.     

Evaluation of MPN method combined with PCR procedure for detection and enumeration of Vibrio parahaemolyticus in seafood

Vibrio parahaemolyticus densities in spiked and naturally contaminated seafood samples were enumerated by the MPN method combined with a PCR procedure (MPN-PCR method) targeting the species-specific thermolabile hemolysin gene (tlh), and by the MPN method using subcultivation of alkaline-peptone-water (APW) enrichment culture on thiosulfate-citrate-bile-sucrose (TCBS) agar (MPN-TCBS method). In the samples spiked with both V. parahaemolyticus and V. alginolyticus, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were similar to, or higher than the numbers of spiked cells, whereas those enumerated by the MPN-TCBS method were below the numbers of spiked cells. In naturally contaminated seafood samples, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were higher than those by the MPN-TCBS method. In the case of the MPN-TCBS method, isolation of V. parahaemolyticus from some APW cultures was difficult because of the overgrowth of many colonies other than V. parahaemolyticus (e.g., V. alginolyticus) on TCBS agar. In contrast, the PCR technique could detect tlh from APW culture without isolation of V. parahaemolyticus, so the possibility of failing to obtain a positive result in APW culture by the MPN-PCR method was considered to be lower than that by the MPN-TCBS method. Furthermore, utilization of the PCR technique reduces the time and labor required for the biochemical identification tests used in the MPN-TCBS method. For the detection and enumeration of V. parahaemolyticus in seafood, especially for samples that show many colonies other than V. parahaemolyticus on TCBS agar, the MPN-PCR method may be more convenient and reliable than the MPN-TCBS method.     

Evaluation of an alkaline phosphatase-labeled oligonucleotide probe for the detection and enumeration of the thermostable-related hemolysin (trh) gene of Vibrio parahaemolyticus

Reliable methods are needed to detect total and pathogenic Vibrio parahaemolyticus. One marker of V. parahaemolyticus virulence is the thermostable-related hemolysin. We developed an alkaline phosphatase-labeled DNA probe method for the specific detection and enumeration of trh-positive V. parahaemolyticus by colony hybridization. The probe was tested against a panel of 200 bacterial strains and determined to be specific for trh-positive V. parahaemolyticus. Additionally, the trh alkaline phosphatase probe colony hybridization was successfully used to detect and enumerate trh-positive V. parahaemolyticus in seafood and water samples collected from the United States and the United Kingdom.     

Development of monoclonal antibody based sandwich ELISA for the rapid detection of pathogenic Vibrio parahaemolyticus in seafood

Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are considered important virulence factors of Vibrio parahaemolyticus and strains producing either of these or both are considered pathogenic. In this study, we generated monoclonal antibodies (mAbs) against purified TRH recombinant protein of pathogenic V. parahaemolyticus. Sandwich enzyme-linked immunosorbent assays (ELISA) using the hybridoma clone 4B10 showed higher sensitivity of detection compared to other clones. Using mAb 4B10 based sandwich ELISA, we could detect pathogenic V. parahaemolyticus in 41.18% (14 out of 34) of the seafood samples analyzed. PCR targeting the toxR gene showed the presence of V. parahaemolyticus in 64.7% (22 out of 34) seafood samples. Further, PCR targeting the virulence genes showed that 6 seafood samples harboured the tdh gene while 9 harboured the trh gene indicating the presence of pathogenic V. parahaemolyticus. Our results show that mAb 4B10 sandwich ELISA developed in this study could be used as a rapid method for screening seafood samples for the presence of pathogenic V. parahaemolyticus.     

副溶血性弧菌PMA-LAMP方法的建立

副溶血性弧菌(Vibrio parahemolyticus)是一种常见的食源性致病菌,在海鲜等食物中检出率很高,食用没有煮熟的带菌食物或者腌制品,极易引起食物中毒。目前用以检测副溶血性弧菌的快速检测方法都不能区别死活菌,容易出现假阳性结果。本研究基于DNA结合染料叠氮溴化丙锭(propidium monoazide,PMA),利用荧光染料钙黄绿素(Calcein)的特性,建立了一种实时的可视化环介导等温扩增方法(Loop-mediated isothermal amplification,LAMP),并与q PCR方法进行比较。实时可视化LAMP方法的反应结果,既可以肉眼直接观测,也可以借助荧光分析设备实时监测,并对半定量分析进行初探,为之后向定量分析深入研究打下基础。实验结果表明,实时可视化PMA-LAMP方法的灵敏度可达5.0×102 CFU/m L,模拟食样灵敏度为1.9×102 CFU/m L,与PMA-q PCR方法结果一致。除去前增菌时间,整个检测过程只需2 h,且准确率高,为副溶血性弧菌快速检测的开展提供了有效的技术支持。     

Levels of Vibrio parahaemolyticus and thermostable direct hemolysin gene-positive organisms in retail seafood determined by the most probable number-polymerase chain reaction (MPN-PCR) method

The incidence and levels of Vibrio parahaemolyticus and thermostable direct hemolysin gene (tdh)-positive organisms in retail seafood were determined. The most probable number-polymerase chain reaction (MPN-PCR) method using a PCR procedure targeting the species-specific thermolabile hemolysin gene (tlh) and tdh was used to determine the levels of V. parahaemolyticus and tdh-positive organisms, respectively. In seafood for raw consumption, V. parahaemolyticus was found in four (13.3%) of 30 fish samples, 11 (55.0%) of 20 crustacean samples, and 29 (96.7%) of 30 mollusc samples. Levels of V. parahaemolyticus were below 10(4) MPN/100 g in all fish and crustacean samples tested. However, they were above 10(4) MPN/100 g in 11 (36.7%) of the 30 mollusc samples. In all seafood for raw consumption, the level of tdh-positive organisms was below the limit of detection (< 30 MPN/100 g). In seafood for cooking, V. parahaemolyticus was found in 15 (75.0%) of 20 fish samples, nine (45.0%) of 20 crustacean sample, and 20 (100%) of 20 mollusc samples. Levels of V. parahaemolyticus were above 10(4) MPN/100 g in only three (15.0%) and one (5.0%) of the 20 fish and 20 crustacean samples, respectively. However, they were above 10(4) MPN/100 g in 18 (90.0%) of the 20 mollusc samples. In seven (35.0%) of the 20 mollusc samples, tdh-positive organisms were found and their levels ranged from 3.6x10 to 1.1 x 103 MPN/100 g. From four of seven tdhpositive samples, tdh-positive V. parahaemolyticus was isolated.     

Molecular typing of Vibrio parahaemolyticus isolates from the middle-east coastline of China

The occurrence of outbreaks of Vibrio parahaemolyticus gastroenteritis in China highlights the need for strain characterization and subtyping of this pathogenic species. A total of 56 epidemiologically-unrelated strains of V. parahaemolyticus were isolated from clinical samples, seafood and various environmental sites in the middle-east coastline of China from 2006 to 2008. The isolates were characterized using four molecular typing methods, including ribotyping, enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), pulsed-field gel electrophoresis (PFGE), and sequence analysis of the gyrB gene. Genetic profiles of cluster analysis from these molecular typing tests clearly showed that there were differences in potential pathogenicity among isolates from seafood and its environments. Genetic characterization of two isolates (F13 and QS2) that originated from seafood demonstrated that they were potentially pathogenic. Discriminatory indices of four typing methods for the 56 V. parahaemolyticus isolates were differentiated by Simpson's Index of Diversity. The discriminatory index of ERIC-PCR typing was maximal (D = 0.942), while that of sequence analysis of the gyrB gene was minimal (D = 0.702). The discriminatory ability was greatly enhanced (D = 0.966) when ERIC-PCR was coupled with sequence analysis of the gyrB gene. These results suggest that ERIC-PCR combined with sequence analysis of gyrB gene may be a reliable, rapid typing strategy for V. parahaemolyticus strains.