沙门氏菌invA基因重组质粒标准的构建

目的 研制沙门氏菌invA基因重组质粒,为分子生物学方法快速检测沙门氏菌提供质粒标准。方法 通过PCR扩增目的片段,连接至pMD 18-T载体,转化大肠杆菌DH5α,测序方法证实目的片段已成功重组,荧光定量PCR方法定性检测分析,采用PicoGreen DNA分子荧光定量方法对标准质粒分子进行定值。结果 invA基因目的片段成功重组至pMD 18-T载体上,荧光定量PCR结果显示制备重组质粒标准为沙门氏菌核酸标准,重组质粒标准的浓度为2.9 μg/mL。结论 成功构建沙门氏菌invA基因重组质粒,为快速检测沙门氏菌奠定了基础。     

婴儿配方粉中阪崎肠杆菌荧光定量PCR检测方法的建立

建立奶粉中阪崎肠杆菌荧光定量PCR检测方法,为准确、快速地定量检测阪崎肠杆菌奠定基础。根据阪崎肠杆菌16S rDNA基因序列,设计特异引物和荧光标记探针并合成,用PCR扩增产物克隆的重组质粒作为阳性对照,经紫外分光光度计测定重组质粒浓度．梯度稀释后作为标准品绘制标准曲线,其Ct值与模板浓度有良好的线形关系,相关系数R^2值为：0．997,可以用于阪崎肠杆菌浓度鉴定。用该方法检测阪崎肠杆菌样品21份,阳性4份,阳性检出率为19％,与常规检测方法结果完全符合。本文建立的检测阪崎肠杆菌的荧光定量PCR方法,较常规技术更为简便、快速,有很好的应用前景和研究价值。     

TaqMan-MGB探针实时荧光定量PCR检测克罗诺杆菌MMS基因方法的建立

建立克罗诺杆菌的特异、灵敏的TaqMan-MGB探针实时荧光定量PCR检测方法。方法 根据GenBank公布的克罗诺杆菌MMS基因高保守序列,设计特异引物和TaqMan-MGB探针,建立和优化反应体系,用25种其他常见致病菌评价反应体系的特异性,用克罗诺杆菌MMS基因重组质粒构建实时荧光定量PCR标准曲线,对重组质粒、纯菌和人工模拟污染样本进行灵敏度试验,并与FDA推荐的TaqMan探针实时荧光PCR比较,配对t检验分析两种方法对Ct值和荧光强度的差异。结果 采用TaqMan-MGB探针实时荧光定量PCR检测克罗诺杆菌MMS基因仅需40min,与25种非目标菌无交叉反应,仅对克罗诺杆菌有特异性扩增;所构建方法线性关系良好,相关系数r²=0.999,扩增效率为99.972%,对重组质粒、纯菌、人工模拟污染样品标本的灵敏度分别达10拷贝/反应、3.8和38cfu/ml;与FDA推荐的TaqMan探针实时荧光PCR相比,TaqMan-MGB探针实时荧光定量PCR的Ct值更小、△Rn值更高,灵敏度和分辨率差异均有统计学意义(Ct:t=-14.406,P﹤0.01;△Rn:t=14.230,P﹤0.01)。结论 本研究建立的TaqMan-MGB探针实时荧光定量PCR反应体系能够快速、特异、灵敏地检测克罗诺杆菌MMS基因,可用于婴幼儿奶粉中克罗诺杆菌的快速筛查和鉴定,具有较大的的应用价值和推广价值。     

志贺氏菌实时荧光单引物等温扩增方法的建立及应用

以志贺氏菌ipa H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检测志贺氏菌的方法,反应时间为44 min。通过对4株不同群志贺氏菌和12株其他食源性致病菌进行实时荧光单引物等温扩增检测,结果表明,除4株志贺氏菌外,其他细菌均未扩增出荧光曲线。进一步研究表明,采用普通热裂解法提取DNA,实时荧光检测福氏志贺氏菌DNA的灵敏度为1.16 fg/μL,纯培养菌液的灵敏度为1.3 CFU/m L;对牛奶模拟样品中福氏志贺氏菌的检出限是1.8 CFU/m L。研究结果表明,实时荧光单引物等温扩增检测志贺氏菌灵敏度高、特异性强、耗时短、方法简便。     

量子点免疫标记法检测福氏志贺氏菌

研究基于抗原抗体反应捕获目标菌,结合生物素与亲和素间的特异性相互作用,联合免疫纳米磁珠磁性分离、免疫量子点荧光标记技术,运用荧光检测系统,建立了福氏志贺氏菌的定量检测模型。结果表明,福氏志贺氏菌菌浓度为102CFU/mL~105CFU/mL,相对荧光强度与菌浓度关系为FI=12.78lgN+15.941,呈良好的线性关系,决定系数R2=0.9761。进一步对模型的准确度和精确度进行验证,得到菌落浓度预测值与真实值差异小,检测相对标准偏差为1.8%,表明模型的准确度好,精密度高。该方法可简单、快速（2h）、高效地检测福氏志贺氏菌。     

三种食源性致病菌多重PCR检测方法的建立

目的:建立同时快速检测沙门氏菌、志贺氏菌和肠出血性大肠杆菌O157∶H7的多重PCR方法。方法:根据沙门氏菌的invA基因、志贺氏菌的ipaH基因及肠出血性大肠杆菌O157∶H7的uidA基因设计3对引物,通过单因素实验、L9(34)正交实验优化反应体系,并对多重PCR扩增的敏感性进行分析。结果:3对引物能特异性扩增出495、620、252bp的目的片段;在最优多重PCR反应体系下,多重PCR检测3种致病菌的灵敏度达104CFU/mL;将该法应用于人工污染实验,可在5h内得到准确、稳定的检测结果。结论:该方法操作简单、检测特异性和灵敏度较高,能够实现对沙门氏菌、志贺氏菌和肠出血性大肠杆菌O157∶H7 3种食源性致病菌的快速监控和诊断。      

牛奶β-乳球蛋白质实时荧光定量PCR检测方法的建立

建立一种快速、特异、灵敏的Taqman实时荧光定量PCR(real-time PCR)方法,用于牛奶主要过敏原β-乳球蛋白质的检测。根据Gen Bank登录的牛β-乳球蛋白质的DNA序列设计,合成一对特异性引物和探针。将扩增产物连接到p MD19-T载体上,制备质粒标准品并测序鉴定,10倍梯度稀释含有β-乳球蛋白质基因的重组质粒,进行实时荧光定量PCR扩增,绘制标准曲线,检测该方法的特异性、稳定性、灵敏性,同时将建立的方法用于10种市售食品的检测。成功建立了β-乳球蛋白质的实时荧光定量PCR检测方法,标准曲线的Ct值与模板浓度在3.18×103~3.18×107copies范围内线性关系良好,R2值为0.997 8;检测灵敏度高(318 copies/μL);特异性强,对羊奶、豆浆DNA均无扩增反应;稳定性好,组内、组间的变异系数均在5%以内。对10种食品牛奶过敏原的检测结果与标签相符。表明所建立的实时荧光定量PCR方法可应用于食品中牛奶过敏原β-乳球蛋白质的检测并可推广到其它过敏原的检测。     

定量检测食品中榛果过敏原成分

定量检测食品中过敏原榛果过敏蛋白,构建目的基因的标准品及标准曲线。以榛果的DNA为模板,针对油体蛋白基因设计引物进行PCR扩增,构建质粒,经鉴定测序,用荧光定量PCR制作标准曲线。成功克隆油体蛋白目的基因片段,并以此为标准制作出荧光定量PCR标准曲线,线性关系良好,灵敏度高,检测限低于10个拷贝,特异性强,准确可靠。成功建立荧光PCR方法定量检测食品中过敏原榛果成分,构建油体蛋白基因的标准品及标准曲线。     

实时荧光定量PCR检测食品中常见食源性致病菌

建立一种快速、准确检测食品中常见食源性致病菌的方法。通过对食品样品提取基因组DNA和实时荧光定量PCR(Real-time Fluorescent Quantitative)条件的优化,建立了沙门氏菌、单核细胞增生性李斯特菌、金黄色葡萄球菌、志贺氏菌、蜡样芽孢杆菌和副溶血弧菌这六种食源性致病菌的实时荧光定量PCR检测方法。通过食品样品的检测,同时进行了传统方法验证。研究建立的实时荧光定量PCR鉴定方法具有良好的重复性和准确性,能够2d出具检测报告,具有较强的实际应用价值,可广泛应用于食品快速检测,具有很好的推广应用前景。     

重组酶介导扩增技术快速检测食品中单核细胞增生李斯特菌

目的建立重组酶介导扩增技术(recombinase aided amplification,RAA)快速检测单核细胞增生李斯特菌的方法。方法以单核细胞增生李斯特菌的hlyA基因作为靶序列,设计引物和探针,通过构建含目的基因片段的重组质粒,评价RAA方法的灵敏度,通过检测沙门氏菌核酸、金黄色葡萄球菌核酸、副溶血性弧菌核酸、溶血性链球菌核酸、志贺氏菌核酸基因组DNA,评价其特异性。结果建立的RAA方法可在39℃、20 min内,检测重组质粒中hlyA基因片段,最低可检出的质粒拷贝数为10拷贝/反应,且与沙门氏菌、金黄色葡萄球菌、副溶血性弧菌、溶血性链球菌、志贺氏菌基因组DNA无交叉反应。结论建立的RAA方法具有灵敏度高和特异性强的特点,可用于单核细胞增生李斯特菌的快速检测。     

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.     

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.     

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.     

Analysis of benzo[a]pyrene in spiked fatty foods by second derivative synchronous spectrofluorimetry after microwave-assisted treatment of samples

A simple, rapid and inexpensive method has been developed for the determination of benzo[a     

The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. II: Certification study

This paper describes the second part of a project undertaken to develop certified mussel reference materials for paralytic shellfish poisoning toxins. In the first part two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin and decarbamoyl-saxitoxin in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the certification exercise. Fifteen laboratories participated in this certification study and were asked to measure saxitoxin and decarbamoyl-saxitoxin in rehydrated lyophilized mussel material and in a saxitoxin-enriched mussel material. The participants were allowed to use a method of their choice but with an extraction procedure to be strictly followed. The study included extra experiments to verify the detection limits for both saxitoxin and decarbamoyl-saxitoxin. Most participants (13 of 15) were able to meet all the criteria set for the certification study. Results for saxitoxin.2HCl yielded a certified mass fraction of <0.07 mg/kg in the rehydrated lyophilized mussels. Results obtained for decarbamoyl-saxitoxin.2HCl yielded a certified mass fraction of 1.59+/-0.20 mg/kg. The results for saxitoxin.2HCl in enriched blank mussel yielded a certified mass fraction of 0.48 +/- 0.06 mg/kg. These certified reference materials for paralytic shellfish poisoning toxins in lyophilized mussel material are the first available for laboratories to test their method for accuracy and performance.     

Announcing a new international peer-reviewed journal:Food Science and Human Wellness now accepting manuscript submission in English

<正>We are pleased to announce the launch of a new international peer-reviewed journal-Food Science and Human Wellness,ISSN 2213-4530,which is an open access journal,produced and hosted by Elsevier B.V.on behalf of Beijing Academy of Food Sciences.Food Science and Human Wellness is an international peer-reviewed English journal that provides a forum for the dissemination of the