食品中金黄色葡萄球菌、单增李斯特氏菌和副溶血性弧菌的MPCR法检测

为快速检测食品中的金黄色葡萄球菌(SA)、单增李斯特氏菌(LM)和副溶血性弧菌(VP),根据各菌的相关基因设计引物,分别扩增金黄色葡萄球菌的耐热核酸酶基因-nuc、单增李斯特氏菌溶血素O上的hlyA基因和副溶血性弧菌的热稳定直接溶血素基因-tdh,建立一种MPCR快速检测食品中致病菌的方法,结果表明,三条特异性扩增片段分别为279bp、 243bp和202bp,经DNA测序证明其序列与模板被扩增片段一致.该方法具有良好的灵敏性和特异性.     

食品中副溶血性弧菌toxR和tdh基因双色荧光PCR检测方法的建立

目的 建立对副溶血性弧菌（Vibrio parahaemolyticus）特异性检测toxR（跨膜转录激活蛋白）基因和tdh（热稳定性直接溶血素）毒力基因的Taqman探针双色荧光PCR检测方法。方法 根据副溶血性弧菌toxR基因和tdh基因,分别设计引物和探针,建立Taqman探针双色荧光PCR扩增体系,进行特异性、灵敏度试验;对副溶血性弧菌分离菌株实施检测,了解其tdh基因和tdh基因分布情况。结果 结果表明,副溶血性弧菌标准菌株和3株从食物中毒患者中分离获得的分离株均出现toxR基因和tdh扩增曲线,而溶藻弧菌、单增李斯特菌等31株弧菌属其他菌株和肠杆菌科的菌株未见扩增曲线。从食品中分离的37株副溶血性弧菌分离株均未携带tdh毒力基因。副溶血性弧菌检测灵敏度可达到3.6×102 cfu/mL。结论 该方法可用于同时检测食品中副溶血性弧菌的特异性和毒力基因。     

牡蛎副溶血性弧菌原位LAMP方法的建立

副溶血性弧菌是造成海产品食源性疾病的重要病原菌之一。为精确定位副溶血性弧菌在牡蛎鳃、外套膜、内脏团、闭壳肌中的分布,明确副溶血性弧菌的组织亲嗜性和侵染进程,研究基于环介导等温扩增技术（LAMP）和原位杂交技术,建立牡蛎副溶血性弧菌原位LAMP检测方法。利用该方法发现副溶血性弧菌经鳃组织过滤,在内脏团消化道快速扩增并蔓延至闭壳肌和外套膜等外围组织,属于Ⅳ型发病机制。该方法同步实现对副溶血性弧菌的高灵敏度和精确定位检测,为进一步研究牡蛎应对副溶血性弧菌的免疫反应奠定了基础。     

副溶血性弧菌ERIC-PCR分型及毒力基因检测研究

目的:建立副溶血性弧菌ERIC-PCR分子分型技术,分析副溶血性弧菌标准菌株及分离株基因组DNA ERIC-PCR指纹图谱,并对副溶血性弧菌毒力基因进行检测,以了解不同来源副溶血性弧菌毒力基因携带情况.方法:提取副溶血性弧菌基因组DNA,以肠杆菌基因间共有重复序列(ERIC)为引物进行PCR扩增,PCR产物经琼脂糖凝胶电泳后用凝胶成像分析仪对图谱进行观察分析,并以相似性系数构建聚类图;通过PCR方法对直接耐热溶血素(TDH)和耐热直接相关溶血素(TRH)进行检测.结果:26株副溶血性弧菌均可扩增产生可重复的DNA指纹图谱,ERIC-PCR可将26株菌分为12个型,分辨力指数为0.926;只在临床分离株中检测到TDH基因,而除一株标准菌株外,所有菌株都未检测到TRH基因.结论:研究显示ERIC-PCR可从分子水平对副溶血性弧菌基因组DNA进行快速指纹图谱分析,同时结合毒力基因检测,能够为副溶血性弧菌食物中毒疾病的预防和流行病学调查提供科学依据.     

副溶血性弧菌实时荧光单引物等温扩增方法的建立

以副溶血性弧菌(Vibrio parahaemolyticus)gyr B基因特异序列为靶序列,设计RNA-DNA组合引物和链终止序列,优化反应体系,建立实时荧光单引物等温扩增(Real-time fluorescence single primer isothermal amplification,实时荧光SPIA)检测副溶血性弧菌的方法。实时荧光SPIA在40 min反应时间内,对3株副溶血性弧菌和16株其他食源性致病菌进行实时荧光SPIA检测,结果表明除3株副溶血性弧菌外,其他细菌均未扩增出荧光曲线。进一步研究表明,实时荧光SPIA检测副溶血性弧菌纯培养DNA的灵敏度为8.2 fg/μL,对副溶血性弧菌菌悬液的检测灵敏度为13.5 CFU/m L;对鳕鱼、海蟹、牡蛎和咸鸭蛋等4种模拟样品中副溶血性弧菌的检出限均为14.7 CFU/g。研究结果表明,实时荧光SPIA检测副溶血性弧菌灵敏度高,特异性强,耗时短,方法简便。     

褶牡蛎体内菌落结构分析及创伤弧菌的定量检测

食用被致病菌污染的牡蛎常引起食品安全问题。采用16S rDNA克隆文库法研究褶牡蛎体内的菌落结构,用荧光定量PCR技术对样品体内的副溶血弧菌、沙门氏菌、创伤弧菌和钩端螺旋体进行定性、定量检测。研究结果表明:褶牡蛎体内的优势菌属为螺旋体属和类杆菌属,分别占总菌数的39.21%和23.53%;样品中含有创伤弧菌,含量为(8.48±0.48)×103cfu/g,而副溶血弧菌、沙门氏菌和钩端螺旋体未检测到。     

副溶血性弧菌的温度预测模型

采用预测食品微生物学的方法研究了副溶血性弧菌的生长规律,测定了4～40 ℃副溶血性弧菌的生长曲线,并在此基础上建立了副溶血性弧菌的初级和二级预测模型.结果表明,在10～40 ℃,副溶血性弧菌的生长曲线呈典型的S形,适合用Gompertz和logistic模型拟合;当温度在4～10 ℃时,适合用线性回归方程拟合.副溶血弧菌的温度模型符合Ratkowsky3式,根据预测模型,副溶血弧菌的最低生长温度Tmin为5.5 ℃,最高生长温度Tmax为47.3 ℃,最适生长温度为34.6 ℃.最后,对模型进行了验证.     

创伤弧菌致病性及致病机制研究进展

创伤弧菌广泛存在于海水和牡蛎等海产品中,具有很强的细胞毒性和溶血性,被美国疾病预防控制中心(CDC)列为三大致病性弧菌之一。本文就创伤弧菌在食品中的污染状况,人类感染途径及临床表现,致病性、致病机制和检测方法等进行综述,以期为该菌感染的预防和治疗提供理论依据。     

一株副溶血弧菌噬菌体生物学特性、全基因组特征及其在食品中的应用

目的：分析1株副溶血性弧菌噬菌体474x1的生物学特性、全基因组及在食品中的抑菌效果。方法：以副溶血性弧菌474菌株为宿主菌，从海鲜市场基围虾分离噬菌体474x1，利用透射电镜观察其形态，并绘制一步生长曲线，分析474x1对温度及pH的敏感性。分析474x1全基因组序列，根据474x1末端酶大亚基构建系统进化树。通过测定菌落总数评价噬菌体对虾肉中副溶血弧菌的抑制效果。结果：分离到1株新型的副溶血弧菌噬菌体，命名为474x1，该噬菌体能够裂解23株副溶血弧菌中的19株（19/23=82.61%）。电镜观察474x1具有典型的短尾病毒科病毒形态特征。最佳感染复数（MOI）为0.01，一步生长曲线显示474x1的潜伏期为10 min，裂解量为115 PFU/cell。该噬菌体能够在较大的温度范围（30～60℃）和pH(4～11)范围内维持活性。474x1全基因组长47830 bp，包含69个开放阅读框（open reading frames,ORFs），其中14个具有特定功能的基因。比较基因组学分析474x1与短尾噬菌体科弧菌属噬菌体Vp41s3基因组具有较高同源性，进化分析表明474x1与...     

海口市市售贝类海产品副溶血性弧菌污染调查及耐药和毒力基因研究

目的了解海口市市售贝类海产品中副溶血性弧菌的污染情况、菌株血清学分型情况以及不同来源菌株的耐药情况,分析海口市市售贝类海产品受副溶血性弧菌污染的特点。方法 2014—2016年,按照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》和《国家食品污染和有害因素风险监测工作手册》对五类海产品(白贝、排海、毛蚶、蛏子、芒果螺)进行副溶血性弧菌分离鉴定和血清学分型,采用实时荧光聚合酶链式反应(PCR)进行毒力基因检测,采用K-B法对分离菌株进行相关耐药分析。结果五类海产品样品共157份,其中65份样品检出副溶血性弧菌,白贝的检出率最高,为63.6%(21/33)。所分离的65株副溶血性弧菌主要血清群为O3和O5,完全分型26株,总体分型率为40.0%,其中以O1∶K25为主要血清型。65株菌对氨苄西林普遍耐药(95.4%,62/65),对头孢噻肟的中介率较高(33.8%,22/65),对8种抗生素产生了5种耐药谱。65株分离菌株神奈川试验结果均为阴性,且均未检出与致病性相关的耐热直接溶血素(TDH)及耐热相关溶血素(TRH)。结论海口市市售贝类副溶血性弧菌污染较重,应加强养殖区域海水水质的监测力度,预防副溶血性弧菌感染。     

食品中二氧化硫现场快速检测方法

目的针对目前食品添加剂亚硫酸盐的滥用现象,寻找出可靠的现场快速测定方法,应用于食品中二氧化硫的现场快速检测。方法从市售二氧化硫快速检测试剂盒中筛选出性能较好的两款产品,分别进行线性、稳定性、检出限测试,并选择易滥用亚硫酸盐的陈粮、米粉等10种食品基质进行加标测试,从阳性对照、与国标方法比对、干扰实验等方面进行比较分析。结果两款试剂盒的线性、稳定性、检出限、加标测试结果良好;在实测的10种食品基质中,陈粮、米粉等7种基质的测定效果良好,鱼糜、冻虾基质会出现阳性干扰;但两款二氧化硫快速试剂盒均不适用于烤虾基质的测定,其结果显示假阴性。结论两款试剂盒均能满足大部分食品中二氧化硫现场快速测定的需求。     

苏州市售动物性水产品兽药残留和重金属含量调研分析

目的研究动物性水产品质量及存在的风险点。方法采集苏州地区市售的8类水产品共300批次样品,检测以兽药残留和重金属为主的30个项目,幵对检测结果进行数据分析和评价。结果 300批次水产品,经检验共有33批次不合栺,不合栺率为11.0%;不合栺产品类别为淡水鱼、淡水虾、海水蟹、海水虾4类,其他4类均合栺;主要不合栺项目为重金属镉和兽药残留:孔雀石绿、恩诺沙星(以恩诺沙星与环丙沙星之和计)、呋喃西林代谢物、氧氟沙星。结论重金属超标和兽药残留问题依然是水产品主要的质量问题,但是不同类别水产品存在的情况有所不同,淡水虾、淡水鱼主要存在兽药残留超标问题,海水蟹、海水虾易存在重金属镉超标现象。皮皮虾、梭子蟹镉超标情况较严重,应当引起重视。     

2013~2015年湖北地区虾中硝基呋喃代谢物残留的监测分析

目的 分析湖北省2013年～2015年347份虾产品中硝基呋喃类药物的残留情况,以期为硝基呋喃代谢物的来源和危害分析提供科学依据。方法 采用超高效液相色谱串联质谱法（UPLC-MS/MS）检测湖北9个地区多种虾产品中四种硝基呋喃类代谢物残留量,并用Excel 和SPSS16.0对实验数据进行统计学分析。结果 硝基呋喃类药物的代谢产物的总体检出率为27.09%,检出的代谢物均为氨基脲（SEM）,河虾的检出率最高,为61.54%,其次是基围虾为6.78%;最低是小龙虾为1.38%;对于整体匀浆的带壳河虾样品,检出率高达91.67%,而去壳的河虾虾肉样品没有检出SEM。结论 湖北地区的虾产品中存在不同程度的硝基呋喃污染情况,同时,发现河虾中有内源性的SEM的存在。结合湖北地区居民的膳食消费结果,进行虾中SEM的风险评估,发现虾中SEM暴露对人类健康危害较小。     

Further data on the production of beauvericin, enniatins and fusaproliferin and toxicity to Artemia salina by Fusarium species of Gibberella fujikuroi species complex

The knowledge of toxigenic profiles of fungal plant pathogens is of extreme importance for evaluating the potential toxicity of infected plant products. Ninety-six fungal isolates belonging to 28 species in the Gibberella fujikuroi complex were studied for the production of beauvericin, enniatins and fusaproliferin in rice cultures. Toxin production ranged from 5 to 3000 microg/g for beauvericin, 2 to 131 microg/g for enniatins, and 4 to 440 microg/g for fusaproliferin. Beauvericin was the most common metabolite produced by 16 species followed by fusaproliferin with 11 species and enniatins with 4 species. The production of beauvericin by F. bulbicola, F. denticulatum, F. lactis, F. phyllophilum, F. pseudocircinatum, and F. succisae and fusaproliferin by F. antophilum, F. begoniae, F. bulbicola, F. circinatum, F. concentricum, F. succisae, and F. udum is reported here for the first time. Brine shrimp larvae were most sensitive to culture extracts of F. acutatum (up to 94+/-3%), F. concentricum (up to 99+/-1%), F. denticuatum (up to 100%) and F. sacchari (up to 100%). Toxicity towards brine shrimp was significantly correlated with the beauvericin content of the fungal extracts with few exceptions. These data indicate that beauvericin and fusaproliferin are common metabolites of species of the G. fujikuroi complex and pose a risk for a possible toxin accumulation in their respective host plant products. However, data from the brine shrimp bioassay showed that further toxic metabolites within this complex need to be characterized.     

云南省淡水湖泊鱼虾中多氯联苯食用安全性评估

目的对云南省5个淡水湖泊中鱼虾体内的多氯联苯(polychorinated biphenyl,PCBs)污染水平进行调查研究。方法采用气相色谱-质谱联用仪进行检测分析,根据检测结果对云南省淡水湖泊中的鱼虾体内多氯联苯的含量进行食用安全性评估。结果云南省淡水湖泊中鱼虾体内的多氯联苯检出率为90%,多氯联苯的含量范围为ND～1.16μg/kg,平均含量为0.27μg/kg,低于国家标准限量。云南省淡水湖泊中鱼虾体内的多氯联苯按照世界卫生组织(World Health Organization,WHO)规定的多氯联苯毒性当量(toxic equivalent quantity,TEQ)分布于ND～6.51×10~(-5)μg/kg,平均值为1.21×10~(-5)μg/kg,低于欧盟的行动标准限量。结论云南省居民从云南省淡水湖泊鱼虾体内摄入持久性有机污染物多氯联苯的剂量较低,引起的健康风险较低。     

Characteristics of Aspergillus niger xylanases produced on rice husk and wheat bran in submerged culture and solid‐state fermentation for an applicability proposal

Xylanolytic rich filtrates were obtained by A. niger sp in both submerged and solid‐state culture using rice husk or wheat bran as the only carbon source. Filtrates obtained on rice husk showed the highest activities (~6500 and 5200 U g^?1, respectively). Independent of carbon source, these filtrates were very stable in an acidic pH range (4–7) and mild temperatures, with high half‐life time values (more than 7 h at 50 °C) in the corresponding inactivation kinetic models. Also the effect of different metallic ions and denaturing substances was verified finding that these enzymes are not metaloproteins, and metals as Hg²⁺ and Pb²⁺ caused the greatest loss of xylanolytic activity (not higher than 30%). Xylanases produced by this A. niger strain showed important features that make them potential candidates for applications on human and livestock food industries.     

Production of secondary metabolites by some terverticillate penicillia on carbohydrate-rich and meat substrates

Most terverticillate penicillia isolated from dry-cured meat products are toxigenic, but their ability to produce hazardous metabolites on meat-based substrates is not well known. The production of extrolites by selected terverticillate penicillia isolated from dry-cured ham has been studied on carbohydrate-rich media (malt extract agar, Czapek yeast autolysate agar, rice extract agar, and rice), meat extract triolein salt agar, and ham slices. Chloroform extracts from the selected strains grown on malt extract agar were toxic for the brine shrimp (Artemia salina) larvae and VERO cells at a concentration of 2 mg/ml, but 0.02 mg/ml produced no toxic effect. Analysis by high-pressure liquid chromatography (HPLC) coupled with photodiode array detection (DAD) or with mass spectrometry (MS) and an atmospheric pressure chemical ionization (APCI) source revealed different biologically active metabolites: cyclopiazonic acid and rugulovasine A from Penicillium commune; verrucosidin, anacine, puberuline, verrucofortine, and viridicatols from Penicillium polonicum; arisugacin and viridicatols from Penicillium echinulatum; and compactin and viridicatols from Penicillium solitum. Most of these metabolites, including the amino acid-derived compounds, were produced in the media containing high levels of carbohydrates. High concentrations of nitrogen compounds in the medium does not imply a greater production of the metabolites studied, not even those derived from the amino acids. However, molds growing on dry-cured ham are able to synthesize limited amounts of some secondary metabolites, a fact not previously reported. The combination of HPLC coupled with DAD and MS-APCI was useful for identification of closely related terverticillate Penicillium species from dry-cured ham. These techniques could be used to characterize the risk associated with the potential production of secondary metabolites in cured meats.     

海鲜香辣酱配方优化

本文对海鲜香辣酱配方进行优化研究,在传统工艺制作基础之上,结合现代中餐标准化生产工艺要求,通过感官评价,利用单因素实验与正交实验,对其配方进行优化,最终确定海鲜香辣酱最佳配方：郫县豆瓣酱1500克、对虾肉700克、牡蛎肉600克、白砂糖130克、花生油900克、清水550克。在此条件下,制作的海鲜香辣酱具有鲜香、麻辣的特点。     

湖南省克氏原螯虾及其养殖水环境中Pb分布情况及相关性研究

采用抽样调查方法从湖南省淡水虾养殖基地对克氏原螯虾及养殖环境进行采样,检测Pb含量,对数据进行统计分析。结果显示:湖南省养殖虾、养殖环境中Pb含量均是益阳>岳阳>常德,其中益阳虾Pb超标率最高,为63%。虾头部、尾部和水体沉积物的Pb含量分布存在地区差异(P<0.05);不同地区养殖水体的Pb含量差异无统计学意义(P=0.051)。养殖环境、虾Pb含量与养殖水及水底沉积物间的差异均具有统计学意义(P<0.05);养殖水与沉积物间差异无统计学意义。逐步Logistic回归分析表明,养殖水和水底沉积物中Pb含量是淡水虾Pb超标的独立危险因素(OR_养殖水=1.018,P<0.05;OR_沉积物=1.505,P<0.000 1)。此外,养殖水及水底沉积物的交互作用也影响淡水虾的Pb超标情况(OR_交互=1.015,P<0.000 1)。综上,湖南省淡水虾养殖基地养殖环境出现不同程度的Pb偏高,且克氏原螯虾Pb与养殖水环境Pb存在明显的相关性。但湖南地区淡水虾养殖基地克氏原螯虾食用部分不存在Pb含量偏高情况,可放心食用。     

Effect of intertidal exposure on Vibrio parahaemolyticus levels in Pacific Northwest oysters

Interest in Vibrio parahaemolyticus (Vp) increased in the United States following Vp-associated gastroenteritis outbreaks in 1997 and 1998 involving the West Coast and other areas. The present study evaluated multiple aspects of Vp ecology in the Pacific Northwest with three objectives: (i) to determine the effect of low-tide exposure on Vp levels in oysters, (ii) to determine the relationship between total and pathogenic Vp, and (iii) to examine sediments and aquatic fauna as reservoirs for pathogenic Vp. Samples were collected from intertidal reefs along Hood Canal, Wash., in August 2001. Fecal matter from marine mammals and aquatic birds as well as intestinal contents from bottom-dwelling fish were tested. Total and pathogenic Vp levels in all the samples were enumerated with colony hybridization procedures using DNA probes that targeted the thermolabile direct hemolysin (tlh) and thermostable direct hemolysin (tdh) genes, respectively. The mean Vp densities in oysters were four to eight times greater at maximum exposure than at the corresponding first exposure. While tdh-positive Vp counts were generally < or = 10 CFU/g at first exposure, counts as high as 160 CFU/g were found at maximum exposure. Vp concentrations in sediments were not significantly different from those in oysters at maximum exposure. Pathogenic (tdh positive) Vp was detected in 9 of 42 (21%) oyster samples at maximum exposure, in 5 of 19 (26%) sediment samples, but in 0 of 9 excreta samples. These results demonstrate that summer conditions permit the multiplication of Vp in oysters exposed by a receding tide.