PCR 法检测动物源性食品中布氏弓形菌

弓形菌是一种新型食源性致病菌,其中以布氏弓形菌污染率最高。本研究采用扩增23S rRNA的PCR法特异性检测布氏弓形菌,方法灵敏度可达103 CFU/mL。2株布氏弓形菌均特异性地扩增出了长度为2061 bp的条带;嗜低温弓形菌、斯氏弓形菌、空肠弯曲菌等共18株不同种类的菌株均无扩增产物出现,表明此PCR法能特异性的将布氏弓形菌鉴定到种一级水平。比较实验结果表明,API CAMPY鉴定试剂盒对于布氏弓形菌鉴定仅能到弓形菌属一级水平,本PCR法能实现布氏弓形菌种一级水平的鉴定,用于布氏弓形菌的检测具有优势。55份动物源性食品样品用Johnson-Murano肉汤增菌后用PCR法进行检测,其中5份样品为布氏弓形菌阳性,阳性检出率为9.1%。上述实验结果表明,本方法特异性强、操作简便,节省了检测时间,可用于动物源性食品中布氏弓形菌的快速检测。     

食品中弓形菌16S rRNA特异性扩增检测方法的建立

针对弓形菌16SrRNA基因合成1对引物,通过对聚合酶链式反应(PCR)扩增条件的优化,建立了检测弓形菌的PCR方法。3株弓形菌标准菌株PCR产物测序结果与NCBI上公布的弓形菌16S rRNA基因序列进行比对,比对结果表明3株弓形菌测序结果与NCBI上公布的弓形菌16S rRNA基因序列同源性均在99%以上。3株弓形菌标准菌株均特异性地扩增出了长度为1202bp的片段,其他19株不同种类的菌株均无扩增产物出现。55份食品样品用Johnson-Murano肉汤增菌后用此法进行检测,其中6份样品为弓形菌阳性,阳性率为10.9%。上述实验结果表明,方法特异性强、操作简便,节省了检测时间,可用于食品中弓形菌的快速检测。     

应用gyrA基因聚合酶链反应检测食品中弓形菌

建立了一种快速、敏感、特异检测弓形菌的PCR方法。选取弓形菌gyrA基因序列作为靶序列设计特异性引物Arco/gyrAF和Arco/gyrAR,进行PCR扩增实验。结果弓形菌呈现出一条298 bp的特异性条带,其余非弓形菌均无阳性扩增。灵敏度实验结果表明,该法可以检测到82.6 pg的DNA。经过对16份生鲜鸡肉样品的检测,9份样品中检测到弓形菌,样品阳性率为56.25%。该PCR方法可以高效、灵敏地检测食品中弓形菌,具有很强的实际应用价值。     

Taqman探针荧光PCR法检测食品中的大肠埃希氏菌O145

针对大肠埃希氏菌O145的O抗原基因簇的wck D基因的特异性序列设计引物和Taqman探针,建立检测大肠埃希氏菌O145的荧光PCR方法,对其灵敏度、特异性进行验证,并将其用于食品样品的检测。结果表明,本研究中的方法可实现对大肠埃希氏菌O145的特异性扩增,其它27株非O145大肠埃希氏菌和20株非大肠埃希氏菌细菌的菌株均无扩增;检测的灵敏度可达165拷贝/反应;339份食品样品EC肉汤增菌后用本荧光PCR法进行检测,检出大肠埃希氏菌O145阳性31份,阳性率为9.1%。实验结果表明,本研究成功建立了可用于食品中大肠埃希氏菌O145的Taqman探针荧光PCR方法,食品样品采用EC肉汤增菌24 h、热裂解法提取核酸,增菌后检测所需时间由至少3 d~7 d缩短为仅需2 h~3 h,食品检测全过程仅需约28 h,经证实本方法特异性强、操作简便,为食品中大肠埃希氏菌O145提供了一种的快速检测手段。     

PCR技术检测动物源性食品中沙门菌的应用研究

为快速准确检测动物源性食品中的沙门菌,将建立并优化的沙门菌PCR检测试剂盒应用于动物源性食品的检测,并于国标法进行了比较.对上海地区238份鸡蛋、685份原料牛奶、283份猪肉、314份牛肉和58份虾仁样品的沙门菌检测表明,PCR法的敏感性和特异性均为100%,与国标法的符合率亦为100%,且检测时间仅为2 d,较国标法大为缩短.该方法可用于动物源性食品中沙门菌的检测,并具有快速、特异、敏感等优点。     

石家庄市零售鸡肉样品中弓形杆菌污染调查

目的调查石家庄市零售鸡肉中弓形杆菌的污染情况,为石家庄市弓形杆菌分布特征和防控提供基础数据支持。方法从石家庄市市内四区的菜市场、超市、连锁肉店随机购买零售鸡肉样品90份,应用驱动增强动力双孔滤膜法从样品中分离培养弓形杆菌,利用弓形杆菌多重聚合酶链式反应(PCR)检测及测序的方法对分离菌株进行种属鉴定,利用16S rDNA、rpoB基因测序的方法验证菌种鉴定结果。结果 90份鸡肉样品中,60份样品检出弓形杆菌86株,其中嗜低温弓形杆菌49株、布氏弓形杆菌37株,弓形杆菌检出率为66.67%(60/90)。60份阳性样品中,嗜低温弓形杆菌阳性样品45份(75.00%),布氏弓形杆菌阳性样品35份(58.33%)。嗜低温弓形杆菌和布氏弓形杆菌混合感染阳性样品20份,占比为22.22%(20/90)。不同采样点弓形杆菌检出率有较大差异。结论石家庄市零售鸡肉中弓形杆菌污染较重,嗜低温弓形杆菌污染率高于布氏弓形杆菌,两种弓形杆菌混合污染样品率较高。     

食品中单核细胞增生性李斯特氏菌PCR快速检测

通过扩增hly基因建立检测单核细胞增生性李斯特氏菌(Lm)的PCR方法.该方法具有较强的特异性,35株经传统方法鉴定的菌株PCR结果均为阳性,而其他三种同属异种菌,包括英诺克李斯特氏菌、绵羊李斯特氏菌和威尔斯李斯特氏菌及非李斯特氏菌均未扩增出特异性的片段.PCR方法对上Lm纯培养物的最低检测限为7.3CFU/μl,对模拟污染的生猪肉和蔬菜的检测低限为4CFU/g,牛奶为4CFU/ml.应用该方法对285份食品样品检测,17份样品Lm呈阳性,结果与常规的分离培养方法完全一致.该种方法具有敏感、特异、快速及准确的优点,可用于食品中Lm的快速检测.     

环介导等温扩增法检测动植物源性单核细胞增生李斯特氏菌

目的采用环介导等温扩增技术检测动植物源性单核细胞增生李斯特氏菌。方法在北京市范围内各业态餐厅购买600份凉菜样品为研究对象,以单核细胞增生李斯特氏菌的hlyA基因为目标基因,设计4条特异性引物,使用环比等温扩增法(loop-to-isothermal amplification, LAMP)创建凉菜中单核细胞增生李斯特氏菌快速检测方法并将其应用于鉴定凉菜中分离的单核细胞增生李斯特氏菌。结果通过细菌分离培养法对600份凉菜进行检测,一共检测分离出37株致病菌菌株,其中包括7株单核细胞增生李斯特氏菌、10株沙门氏菌和20株大肠埃希氏菌,单核细胞增生李斯特氏菌的检出率为1.2%。用LAMP对样品中的菌株进行测定,7株凉菜中分离的单核细胞增生李斯特氏菌为阳性,其他菌株为阴性。通过LAMP方法检测动植物源性凉菜中分离的单核细胞增生李斯特氏菌平均检出限为2.2*10 CFU/μL、特异性为100%。动物源性凉菜中的致病菌含量较高。结论环介导等温扩增法具备灵敏、快速、操作简便的特点,适合用于餐饮行业的现场快速检测领域。     

实时荧光PCR法与环介导等温扩增法检测食品中动物源性成分的比较

目的比较实时荧光PCR(real-time PCR)法及环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测食品中动物源性成分,为食品安全监管部门动物源性成分鉴定提供准确、高效的检测方法。方法采用实时荧光PCR法及LAMP法分别对市售肉及其制品进行牛、猪和鸡源性成分鉴定,并与标签明示的肉源成分比对,以准确性和时效性2个指标对上述2个方法进行评价。结果 Real-time PCR法检出4份样品与标签明示肉源不符,LAMP法检出5份样品与标签明示肉源不符,而16份样品中仅有1份样品2种方法检测结果不同。Real-time PCR法检测用时1.5 h,提取检测用DNA用时1.5 h,总用时3 h;而LAMP法检测用时45 min,提取检测用DNA用时20 min,总用时65 min。结论 LAMP法与Real-time PCR均具有较好的特异性、准确性,但LAMP法耗时短、成本低、操作简单,便于现场快速监督抽检。     

环介导等温扩增方法在食品中沙门菌检测的应用和评价

将环介导等温扩增检测方法应用于食品中沙门菌的检验,并在检测方法特异性、灵敏度等方面与实时荧光PCR和传统检测方法进行比较。方法 针对沙门菌属高度保守的fimY基因设计环介导等温扩增检测引物并优化反应体系,在特异性、灵敏度和实际样品检测等方面与实时荧光PCR及传统检测方法比对。结果 本研究建立的LAMP方法检测沙门菌93株和非目标菌31株,具有良好的特异性。在纯培养、无需增菌情况下,其检测灵敏度为6.4×10²cfu/ml,与实时荧光PCR方法相当。食品基质添加试验中,环介导等温扩增方法检测低限为2cfu/25g样品;对45份实际食品样品检测结果表明,该方法实际样品检出率为11.1%,与实时荧光PCR及传统方法检测结果一致。结论 本研究建立的沙门菌环介导等温扩增检测方法具有良好的特异性,检测灵敏度与实时荧光PCR相当,适用于沙门菌的快速筛选。     

Reliable identification of grapevine rootstock varieties using RAPD PCR on woody samples

Since grapevine ( Vitis spp .) rootstock material is being traded increasingly as disbudded woody material a lack of distinctive morphological features on such material necessitates an alternative and reliable means of identification. Methods described here were developed for rapid and efficient extraction of DNA from woody samples rich in phenolic compounds and polysaccharides, and for subsequent identification of varieties by RAPD PCR. Using these methods, and with the application of only one selected RAPD primer, we were able to differentiate sixteen rootstock varieties, including the seven varieties most commonly used in Germany. Problems commonly encountered with reproducibility of RAPD patterns were avoided by choosing primers with a dinucleotide sequence and a high G/C content that allowed a rather high annealing temperature of 45°C. Methods described here should also be useful for other horticultural crops, especially those with woody tissues rich in phenolic compounds and polysaccharides.     

An internet compendium of analytical methods and spectroscopic information for monomers and additives used in food packaging plastics

An internet website (http://cpf.jrc.it/smt/) has been produced as a means of dissemination of methods of analysis and supporting spectroscopic information on monomers and additives used for food contact materials (principally packaging). The site which is aimed primarily at assisting food control laboratories in the European Union contains analytical information on monomers, starting substances and additives used in the manufacture of plastics materials. A searchable index is provided giving PM and CAS numbers for each of 255 substances. For each substance a data sheet gives regulatory information, chemical structures, physico-chemical information and background information on the use of the substance in particular plastics, and the food packaging applications. For monomers and starting substances (155 compounds) the infra-red and mass spectra are provided, and for additives (100 compounds); additionally proton NMR are available for about 50% of the entries. Where analytical methods have been developed for determining these substances as residual amounts in plastics or as trace amounts in food simulants these methods are also on the website. All information is provided in portable document file (PDF) format which means that high quality copies can be readily printed, using freely available Adobe Acrobat Reader software. The website will in future be maintained and up-dated by the European Commission's Joint Research Centre (JRC) as new substances are authorized for use by the European Commission (DG-ENTR formerly DGIII). Where analytical laboratories (food control or other) require reference substances these can be obtained free-ofcharge from a reference collection housed at the JRC and maintained in conjunction with this website compendium.     

Chlorohydrins of bisphenol A diglycidyl ether (BADGE) and of bisphenol F diglycidyl ether (BFDGE) in canned foods and ready-to-drink coffees from the Japanese market

BADGE.2HCl and BFDGE.2HCl were determined in 28 samples of ready-to-drink canned coffee and 18 samples of canned vegetables (10 corn, 5 tomatoes and 3 others), all from the Japanese market. HPLC was used as the principal analytical method and GCMS for confirmation of relevant LC fractions. BADGE.2HCl was found to be present in one canned coffee and five samples of corn, BFDGE.2HCl in four samples of canned tomatoes and in one canned corn. No sample was found which exceeded the 1mg/kg limit of the EU for the BADGE chlorohydrins. However the highest concentration was found for the sum of BFDGE.2HCl and BFDGE.HCl.H₂O at a level of 1.5mg/kg. A Beilstein test confirmed that all cans containing foods contaminated with BADGE.2HCl or BFDGE.2HCl had at lest one part coated with a PVC organosol.     

European priorities for research to support legislation in the area of food contact materials and articles

A strong science base is required to underpin the planning and decision-making process involved in determining future European community legislation on materials and articles in contact with food. Significant progress has been made in the past 5 years in European funded work in this area, with many developments contributing to a much better understanding of the migration process, and better and simpler approaches to food control. In this paper this progress is reviewed against previously identified work-areas (identified in 1994) and conclusions are reached about future requirements for R&D to support legislation on food contact materials and articles over the next 5 or so years.     

Aroma characterization of various apricot varieties using headspace–solid phase microextraction combined with gas chromatography–mass spectrometry and gas chromatography–olfactometry

The characterization of the aromatic profile of several apricot cultivars with molecular tracers in order to obtain objective data concerning the aromatic quality of this fruit was undertaken using headspace–solid phase microextraction (HS–SPME). Six apricot cultivars were selected according to their organoleptic characteristics: Iranien, Orangered, Goldrich, Hargrand, Rouge du Roussillon and A4025. The aromatic intensity of these varieties measured by HS–SPME–Olfactometry were defined and classified according to the presence and the intensity of grassy, fruity and apricot like notes. In the six varieties, 23 common volatile compounds were identified by HS–SPME–GC–MS. Finally, 10 compounds, ethyl acetate, hexyl acetate, limonene, β-cyclocitral, γ-decalactone, 6-methyl-5-hepten-2-one, linalool, β-ionone, menthone and (E)-hexen-2-al were recognized by HS–SPME–GC–O as responsible of the aromatic notes involved in apricot aroma and considered as molecular tracers of apricot aromatic quality which could be utilized to discriminate apricot varieties.     

Tests of potential functional barriers for laminated multilayer food packages. Part I: Low molecular weight permeants

The advent of the functional barrier concept in food packaging has brought with it a requirement for fast tests of permeation through potential barrier materials. In such tests it would be convenient for both foodstuffs and materials below the functional barrier (sub-barrier materials) to be represented by standard simulants. By means of inverse gas chromatography, liquid paraffin spiked with appropriate permeants was considered as a potential simulant of sub-barrier materials based on polypropylene (PP) or similar polyolefins. Experiments were performed to characterize the kinetics of the permeation of low molecular weight model permeants (octene, toluene and isopropanol) from liquid paraffin, through a surrogate potential functional barrier (25 μm-thick oriented PP) into the food simulants olive oil and 3% (w/v) acetic acid. These permeation results were interpreted in terms of three permeation kinetic models regarding the solubility of a particular model permeant in the post-barrier medium (i.e. the food simulant). The results obtained justify the development and evaluation of liquid sub-barrier simulants that would allow flexible yet rigorous testing of new laminated multilayer packaging materials.     

Textural Properties of Cold-set Gels Induced from Heat-denatured Whey Protein Isolates

A 9% whey protein (WP) isolate solution at pH 7.0 was heat-denatured at 80°C for 30 min. Size-exclusion HPLC showed that native WP formed soluble aggregates after heat-treatment. Additions of CaCl2 (10–40 mM), NaCl (50–400 mM) or glucono-delta-lactone (GDL, 0.4–2.0%, w/v) or hydrolysis by a protease from Bacillus licheniformis caused gelation of the denatured solution at 45°C. Textural parameters, hardness, adhesiveness, and cohesiveness of the gels so formed changed markedly with concentration of added salts or pH by added GDL. Maximum gel hardness occurred at 200 mM NaCl or pH 4.7. Increasing CaCl2 concentration continuously increased gel hardness. Generally, GDL-induced gels were harder than salt-induced gels, and much harder than the protease-induced gel.     

Influence des ions sur le pouvoir hydratant de l'ure e: e tude sur peau de porc ex vivo

This study deals with the influence of ions (NaCl and MgSO4) in a W/O emulsion containing 10% urea. Moisturization kinetics are assessed by corneometry on pig skin ex vivo. The formula's influence on urea penetration is measured by infrared spectrometry with an ATR device and the stripping method. Corneometry and spectroscopy were chosen to record simultaneously the hydratation levels and urea localization into superficial cell layers. Urea crystallization after evaporation of emulsions and aqueous solutions is described. Results show that urea does not hydrate nor penetrate when applied to the skin through an aqueous gel. In a W/O emulsion, sodium chloride increases the ability of urea to moisturize without improving penetration. In vitro urea crystallization is disturbed by sodium chloride or magnesium sulphate for solutions and emulsions. This stabilization by ions is correlated with good moisturization values. The stabilization of urea in the solute state provided by ions increases its water epidermal binding capacity without enhancing penetration.     

Occurrence of bisphenol-F-diglycidyl ether (BFDGE) in fish canned in oil

The levels of bisphenol-F-diglycidyl ether (BFDGE) were quantified as part of a European survey on the migration of residues of epoxy resins into oil from canned fish. The contents of BFDGE in cans, lids and fish collected from all 15 Member States of the European Union and Switzerland were analysed in 382 samples. Cans and lids were separately extracted with acetonitrile. The extraction from fish was carried out with hexane followed by re-extraction with acetonitrile. The analysis was performed by reverse phase HPL C with fluorescence detection. BFDGE could be detected in 12% of the fish, 24% of the cans and 18% of the lids. Only 3% of the fish contained BFDGE in concentrations considerably above 1mg/kg. In addition to the presented data, a comparison was made with the levels of BADGE (bisphenol-A-diglycidyl ether)analysed in the same products in the context of a previous study.