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.     

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。     

基于内参的副溶血性弧菌实时荧光定量PCR快速检测方法的建立

目的建立基于内参的副溶血性弧菌实时荧光定量PCR方法,快速检测样品中的副溶血性弧菌。方法根据Gen Bank已公布的副溶血性弧菌基因组序列,筛选特异性靶基因,设计特异性引物探针,优化反应体系,并在体系中加入内参(IAC),通过标记不同荧光基团的Taq Man探针来监测IAC,进而实时监控整个PCR反应。按照5~50 cfu/25 g的细菌量人工污染样品,以评价所建立反应的体系。结果以副溶血性弧菌基因组DNA为模板,最低检测限为1 pg/μl;以10倍梯度稀释的菌液经水煮法提取的DNA为模板,最低检测限为4×102cfu/ml;以含有gyr B的质粒为模板,最低检测极限可以达到100 copies/μl;建立gyr B和gyr B-IAC标准曲线,Ct值与模板拷贝数均呈良好线性关系(r2=0.999);人工污染初始菌量为7 cfu/25 g时,样品中副溶血性弧菌增菌6 h即可检出。结论本研究所建立的gyr B-IAC实时荧光定量PCR方法,既能有效检测食品中副溶血性弧菌,又能实时监测PCR反应过程,有效防止"假阴性"的发生,结果可靠,有利于实现海产品中副溶血性弧菌实时荧光定量PCR检测方法的标准化。     

模拟水产品中副溶血性弧菌定量质控品的制备

目的:制备模拟水产品中副溶血性弧菌(Vibrio parahaemolyticus,VP)定量质控品,并对检测结果进行分析,为微生物质控定量检测工作的开展提供依据。方法:以奶粉为基质,添加一定量的菌液,制备不同的样品A、B、C。样品真空干燥后,于4℃条件下保存,分别于不同时间对样品进行抽查检测。利用复现性临界差值(CD)对样品进行评价。结果:副溶血弧菌计数结果 100%在控制范围内。结论:建立了有效的模拟水产品中副溶血性弧菌定量检测质控品制备的方法及评价程序。     

水产品中副溶血性弧菌特异性二重PCR检测方法的研究

建立快速检测水产品中副溶血性弧菌(Vibrio parahae-molyticus)的二重PCR方法.以副溶血性弧菌特异性基因tlh和toxR为靶基因,选择2对引物,对5株副溶血性孤菌和40株非副溶血性弧菌进行特异性检测;梯度稀释副溶血性弧菌基因组DNA,以不同稀释度DNA作PCR扩增;在鱼肉样品中以不同菌量人工污染,不同增菌时间培养,提取DNA进行PCR扩增;应用该方法对实际样品进行检测.以tlh和toxR为靶基因的两对引物对副溶血性弧菌的检出有很好的特异性.PCR检测的灵敏度在DNA水平上达到28.76 pg;人工污染样品,当起始污染量为1 CFU/mL时,37℃增菌培养10 h即可检出.本试验一共检测了21份水产品样品,有14份检出了副溶血性弧菌.     

纳米免疫磁分离-实时荧光聚合酶链式反应快速检测海产品中副溶血性弧菌

采用副溶血性弧菌单克隆抗体、纳米免疫磁分离技术,结合实时荧光聚合酶链式反应（polymerase chain reaction,PCR）检测方法,建立海产品中副溶血性弧菌纳米免疫磁分离-实时荧光PCR检测方法。副溶血性弧菌纳米免疫磁珠在菌体浓度为103 CFU/mL水平时,对副溶血性弧菌的捕获率达到74%。在纯培养、无需增菌情况下,该方法检测灵敏度达到140 CFU/mL;通过134 株副溶血性弧菌和74 株非目标菌的测试,实时荧光PCR技术具有良好的特异性;在食品基质添加实验中,其检测限为2 CFU/25 g样品,增菌时间缩短到8 h。     

烟台海域海产品中食源性致病菌污染状况调查及膳食风险分析

目的了解烟台濒临的黄海和渤海海域海产品中食源性致病菌污染分布特征,掌握致病菌污染的"基线值",为市场监管、消费指导和风险预警提供数据支持。方法按照GB 4789规定的方法,进行6种食源性致病菌检测。借助快速微生物定量风险评估(s QMRA)方法,评价海产品中副溶血性弧菌的感染风险。结果 6类260种海产品中仅有副溶血性弧菌阳性检出,创伤弧菌、金黄色葡萄球菌、沙门菌、单增李斯特菌和大肠杆菌O157:H7均未检出。海产品中副溶血性弧菌总体污染率为19.62%(51/260),贝类、甲壳类污染水平较高,鱼类、海藻类偏低,污染率分别为26.42%(28/106)、20.00%(6/30)、10.00%(3/30)、10.00%(3/30);贝类中牡蛎是副溶血性弧菌高污染的海产品,污染率为31.03%(9/29)。普通人群摄食加热海产品后副溶血性弧菌致病风险概率值为2.97×10-7,年均患病率为6.03×10~(-6)次/人年,7~9月份为高发病时间段。结论烟台海域鲜活海产品主要存在副溶血性弧菌的污染,摄食人群具有潜在的感染风险,尤其温度较高的第三季度。     

2017年广西壮族自治区5个城市贝类海产品致病性弧菌污染状况分析

目的 了解广西壮族自治区贝类海产品中副溶血性弧菌、创伤弧菌、溶藻弧菌和霍乱弧菌的污染现状,为食源性疾病防控和微生物风险评估提供参考依据。方法 2017年在广西壮族自治区3个沿海和2个内陆城市采集贝类海产品进行定性检测,其中副溶血性弧菌同时进行定量检测和毒力基因检测。结果 5个城市共采集800份贝类海产品,致病性弧菌总阳性率为76.5%(612/800),副溶血性弧菌、创伤弧菌和霍乱弧菌阳性率分别为73.9%(591/800)、18.4%(147/800)、0.1%(1/800),未检出溶藻弧菌。副溶血性弧菌阳性率与采样地区、样品状态和贝类品种有关,沿海地区阳性率高于内陆地区,但含量却低于内陆地区;活产品阳性率和含量均高于鲜/冰鲜海产品;蛏子、泥蚶、牡蛎和蛤/蚬子阳性率较高,均在75.0%以上,扇贝和贻贝阳性率相对较低,但含量较高;1.0%(6/591)的菌株检出致病性毒力基因。创伤弧菌阳性率与样品来源、采样地区和贝类品种有关,沿海地区高于内陆地区,农村高于城市,蛏子和泥蚶阳性率最高,均在35.0%以上。结论 广西壮族自治区贝类海产品中副溶血性弧菌和创伤弧菌污染较严重,需重点加强贝类海产品食品安全卫生宣教,加强沿海农村地区创伤弧菌监测。     

上海市售海产品中副溶血性弧菌的分布状况

海产品中副溶血性弧菌引起的食物中毒已成为我国沿海地区细菌性食物中毒的首要病原。本研究中针对上海市闵行区某农贸市场的海产品开展为期1年的调查分析,共采集257份样品。按照国家标准(GB/T4789.7-2008),以硫代硫酸钠柠檬酸胆盐蔗糖培养基(TCBS)和科玛嘉弧菌显色培养基(CV)两种选择性培养基辅助分离副溶血性弧菌疑似菌株,结合生化试验和PCR方法对疑似菌株进行鉴定,从84份阳性样本中获得107株副溶血性弧菌分离株。其结果表明:该农贸市场市售海产品中副溶血性弧菌的污染率为32.7%,其中牡蛎中副溶血性弧菌污染率最高(达54.4%),蛤蜊和海瓜子次之(分别为33.9%,25.6%)。牡蛎中副溶血性弧菌的污染水平与季节性变化直接相关。对107株分离株的主要毒力基因tdh和trh进行PCR筛查,tdh阳性菌株为10株,trh阳性菌株为1株,并且此株菌为tdh、trh双阳性菌株,tdh和trh的携带率分别为9.4%和1.0%,tdh、trh双基因的携带率为1.0%。结论:市售海产品中副溶血性弧菌污染状况较为严重。这为政府相关职能部门开展食品安全防控提供了参考依据。     

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。     

Characteristics of a sharp decrease in Vibrio parahaemolyticus infections and seafood contamination in Japan

Vibrio parahaemolyticus has been one of the most important foodborne pathogens in Japan since the 1960s, and a large epidemic was caused by the pandemic serotype O3:K6 from 1997 to 2001. V. parahaemolyticus infections, however, have sharply declined since that time. Data on serotypes isolated from 977 outbreaks were collected and analysed. Total and pathogenic, thermostable direct hemolysin (TDH) gene-positive V. parahaemolyticus were qualitatively and quantitatively detected in 842 seafood samples from wholesale markets in 2007-2009. Strains isolated from patients and seafood were analysed by serotyping, tdh-PCR, group-specific PCR for pandemic strains, and pulsed-field gel electrophoresis (PFGE). The sharp decrease in the infections from 1999 onwards was noted not only for O3:K6 infections but also for other serotypes. The change in the seafood contamination situation from 2001 to 2007-2009 was characterised by a decrease to three-fourths in the frequency of tdh-positive samples, although that decrease was small compared to the 18-fold decrease in the cases of V. parahaemolyticus outbreaks. PFGE detected the pandemic O3:K6 serotype in the same profile in seafood and patients from 1998 to the present. Because of no large decrease in seafood contamination by V. parahaemolyticus from the production to distribution stages and the presence of pandemic O3:K6 serotype in seafood to the present, it was suggested that the change of seafood contamination was unrelated to the sharp decrease in V. parahaemolyticus infections. V. parahaemolyticus infections might be prevented at the stages after the distribution stage.     

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.     

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.     

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 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.     

2003年中国部分沿海地区零售海产品中副溶血性弧菌污染状况的主动监测

国家食源性疾病监测网发现，我国近年来副溶血性弧菌中毒呈显上升趋势。为进一步了解零售海产品中副溶血性弧菌(VP)的污染情况，2003年9-12月在我国沿海4个省份(浙江、江苏、广东、福建)进行监测，试样分别从水产品批发市场、零售市场和饭店采集，共采集海产品236份，其中甲壳类69份、贝类116份、鱼类51份。采用Vitek鉴定系统和最可能数(MPN)法进行副溶血性弧菌的定性和定量分析。结果显示，38．6％的海产品检出VP，浙江省试样的VP阳性率最高。甲壳类、贝类和鱼类试样VP阳性率分别为49．3％、37．9％和25.5％；阳性试样几何平均分布浓度依次为171．4、76．9和50．7MPN／100g。监测结果表明，我国零售海产品中副溶血性弧菌的污染率较高，必须持续地进行食品中VP的主动监测和污染控制。     

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。     

Seasonal distribution of total and pathogenic Vibrio parahaemolyticus in Chesapeake Bay oysters and waters

The objectives of this study were to investigate the seasonal distribution of total and pathogenic Vibrio parahaemolyticus in the Chesapeake Bay oysters and waters, and to determine the degree of association between V. parahaemolyticus densities and selected environmental parameters. Oyster and water samples were collected monthly from three sites in Chesapeake Bay, Maryland from November 2004 through October 2005. During collection of samples, water temperature, salinity, turbidity, dissolved oxygen, pH, chlorophyll a, and fecal coliform levels in oysters were also determined. V. parahaemolyticus levels were enumerated by a quantitative direct-plating method followed by DNA colony hybridization; presence/absence was further determined by overnight broth enrichment followed by either standard colony isolation or real-time PCR. The thermolabile hemolysin (tlh) gene and thermostable direct hemolysin (tdh) gene were targeted for detection of total and pathogenic V. parahaemolyticus, respectively, for both direct plating and enrichment. The thermostable related hemolysin (trh) gene, which is a presumptive pathogenicity marker, was targeted only for the enrichment approach. By direct plating, colonies producing tlh signals were detected in 79% of oyster samples at densities ranging from 1.5x10(1) to 6.0x10(2) CFU/g. Pathogenic V. parahaemolyticus (tdh+) was detected in 3% (level was 10 CFU/g) of oyster samples while no V. parahaemolyticus was detected in water samples. By the enrichment approach with standard colony isolation, 67% of oyster and 55% of water samples (n=33) were positive for total V. parahaemolyticus, and all samples were negative for pathogenic V. parahaemolyticus. In contrast, enrichment followed by real-time PCR detected tlh, tdh and trh in 100%, 20% and 40% of oyster and 100%, 13% and 40% of water enrichments collected from June to October 2005, respectively. V. parahaemolyticus densities in oysters varied seasonally and were found to be positively correlated with water temperature, turbidity, and dissolved oxygen.     

东南沿海地区零售海产品中创伤弧菌的监测

目的了解我国东南沿海地区零售海产品中创伤弧菌的污染状况。方法采用3%氯化钠碱性蛋白胨水增菌,改良纤维二糖-多粘菌素B-多粘菌素E(mCPC)琼脂和纤维二糖-多粘菌素E(CC)琼脂分离海产品试样中的创伤弧菌,可疑菌落的鉴定采用生化试验或PCR法。采用最可能数(MPN)法和PCR法检测牡蛎试样的创伤弧菌污染水平。结果天然污染海产品试样创伤弧菌的检出率为19.8%(20/101)。牡蛎试样中45%(55/122)创伤弧菌密度低于3MPN/g的最低检出限。牡蛎中创伤弧菌的污染水平存在季节性差异。结论未来应加强对我国海产品中创伤弧菌污染状况的监测,尤其是在温暖的季节。