葡萄球菌A型肠毒素检测竞争ELISA方法的建立

为建立快速、灵敏、特异的检测食品中金黄色葡萄球菌A型肠毒素的方法,应用构建的pGEX-6P-SEA、pET28α-SEA重组质粒表达带有不同融合肽的葡萄球菌A型肠毒素,分别通过谷胱甘肽亲和纯化、镍螯合纯化获得SEA的重组融合表达蛋白HIS-SEA、GST-SEA,以HIS-SEA为包被抗原,以GST-SEA、天然SEA肠毒素为竞争抗原,确立了SEA型肠毒素ELISA检测的工作条件:包被浓度为2.5μg/mL,阳性血清稀释度为1∶50,辣根过氧化物酶标记的羊抗鸡免疫球蛋白工作浓度为1∶2000。建立了以重组肠毒素GST-SEA作为竞争抗原检测葡萄球菌肠毒素SEA的间接竞争ELISA（ciELISA）方法,竞争肠毒素GST-SEA蛋白浓度x与P/N值y的关系式:x（GST-SEA）=1.99-y/0.34,R2（GST-SEA）=0.9964,线性检测范围均为1～62.5ng/mL,最低检测量为1ng/mL,此方法丰富了食品中A型肠毒素的检测手段。      

胶体金免疫层析技术快速定量检测金黄色葡萄球菌肠毒素B

目的建立胶体金免疫层析技术快速定量检测金黄色葡萄球菌肠毒素B的方法。方法利用胶体金标记和双抗体夹心免疫层析技术,建立金黄色葡萄球菌肠毒素B的快速检测方法,评价其特异性和敏感性,并拟合检测曲线进行定量检测;在牛奶样品中添加金黄色葡萄球菌肠毒素B作为模拟污染样品进行检测。结果该法可在5～10min内完成定性和半定量检测,检出限为8ng/ml,线性范围8～1000ng/ml。结论建立的检测金黄色葡萄球菌肠毒素B的胶体金免疫层析方法,能快速、灵敏、特异、准确地检测样品中的金黄色葡萄球菌肠毒素B,并可实现定量,适用于现场快速检测。     

乳源性金黄色葡萄球菌肠毒素A的间接ELISA检测方法建立

从乳品中筛选出金黄色葡萄球菌,并对其所产的肠毒素进行分离纯化。制备出金黄色葡萄球菌肠毒素的特异性抗血清,建立金黄色葡萄球菌肠毒素A特异性血清间接ELISA方法。结果表明,最佳抗原包被浓度为10μg/mL,抗体最佳稀释倍数为1︰1 600,抗血清效价达到1︰3 200。通过建立此检测方法可以为乳源金黄色葡萄球菌肠毒素的检验提供依据。     

建立金黄色葡萄球菌肠毒素B悬浮芯片定量检测方法

目的建立金黄色葡萄球菌肠毒素B悬浮芯片定量检测方法。方法利用双抗体夹心法免疫学原理,以金黄色葡萄球菌肠毒素B的特异性抗体包覆微球为载体,利用悬浮芯片(Bio-Plex)系统建立检测模型;检测不同浓度金黄色葡萄球菌肠毒素B,测定方法的灵敏性;通过该方法对大肠杆菌、普通变型杆菌、蓖麻毒素和禽流感病毒HA、NH蛋白和金黄色葡萄球菌热休克毒素的检测,判断方法的特异性;将不同浓度金黄色葡萄球菌肠毒素B添加到奶粉中验证方法的实用性和稳定性。结果悬浮芯片方法对金黄色葡萄球菌肠毒素B的检测线性范围为0.2～1653.4ng/ml,最低检测值为203pg/ml,均优于酶联免疫吸附实验;除与2.3μg/ml金黄色葡萄球菌热休克毒素有交叉反应外,和其他几种细菌、毒素、蛋白均无交叉反应;对添加相同浓度金黄色葡萄球菌肠毒素B奶粉的检测的相对标准偏差在1.30%～16.93%。结论悬浮芯片定量检测方法对于模拟添加的金黄色葡萄球菌肠毒素B具有良好的检测效果。     

利用简并引物检测金黄色葡萄球菌肠毒素A、B基因

通过设计简并引物建立一种PCR技术同步快速检测金黄色葡萄球菌肠毒素A和B基因的方法。根据金黄色葡萄球菌肠毒素A、B基因编码序列,设计一对特异性简并引物SEAB来扩增靶基因片段,长度分别为105bp和135bp,通过对金黄色葡萄球菌肠毒素A、B菌株和4株对照菌株进行PCR检测,评价该引物的特异性;对金黄色葡萄球菌肠毒素B的DNA系列10倍稀释,对其灵敏性进行PCR检测。结果显示,金黄色葡萄球菌肠毒素A和B菌株的DNA检测结果呈阳性,4株对照菌株的检测结果呈阴性,通过基因测序证实了PCR产物的特异性,SEB的DNA最低检测浓度为3.58ng,整个检测过程不超过20h。建立了一个特异、快速灵敏的在同一条件下,金黄色葡萄球菌肠毒素A、B基因的PCR检测方法。     

基于纳米抗体的酶联免疫吸附法检测食品中金黄色葡萄球菌肠毒素B

在前期已获得抗金黄色葡萄球菌肠毒素B (Staphylococcal enterotoxin B,SEB)纳米抗体的基础上,建立一种用于检测食品中SEB的酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA),为食品及农产品质量监管提供技术支持。优化表达条件后,通过大肠杆菌原核表达获得抗SEB的纳米抗体B7;以纳米抗体B7作为捕获抗体,以噬菌体展示的抗SEB纳米抗体B6为检测识别元件,建立夹心ELISA方法;以牛奶、牛肉及西瓜汁为样本对其进行方法学评价。结果显示,纳米抗体B7在大肠杆菌中成功表达,表达量为6. 2 mg/L;该方法在16～1 024 ng/m L具有良好的线性关系(R2=0. 990 4),最低检出限为(9. 58±0. 07) ng/m L;该方法与SEC有42. 18%的交叉,与SEA及另外3株金黄色葡萄球菌无明显交叉;牛奶、牛肉和西瓜汁的加标回收率分别为87. 26%～108. 43%,79. 36%～106. 56%和83. 81%～99. 43%。该方法以纳米抗体作为识别元件,可应用于实际食品及农产品中的SEB高灵敏检测,具有广阔的应用前景。     

竞争抑制酶联免疫吸附法快速检测金黄色葡萄球菌肠毒素P

目的 建立一种稳定性强、灵敏度高的快速检测金黄色葡萄球菌新型肠毒素P(staphylococcal enterotoxin P,SEP)的单克隆抗体竞争抑制酶联免疫吸附法(Enzyme-linked Immunosorbent assay,ELISA)。方法 根据ELISA的检测程序,利用交叉方阵滴定法确定SEP抗原的最佳包被浓度及单克隆抗体的最佳倍比稀释浓度,再对辣根过氧化物酶标记的羊抗鼠IgG(HRP-IgG)最佳稀释浓度及最佳反应时间进行筛选,然后通过测定450nm处不同SEP抗原包被条件(包被液类型、包被环境)、封闭液类型及浓度、封闭时间、竞争反应温度、反应方式(预混反应、直接反应)下的OD值对检测条件进行优化,最后用灵敏度、批内变异、批间变异和加标样品回收率对方法进行评价。结果 SEP抗原最佳包被浓度为2.5 μg/mL,鼠单抗血清稀释度1:6000,酶标抗稀释度1:3000;反应1 h,最佳包被条件为磷酸盐缓冲液4 ℃过夜,10%脱脂乳封闭2 h,37 ℃直接竞争反应。此方法的线性回归方程为：y = 0.4166x - 0.7415(R2 = 0.9908),灵敏度为0.954 μg/mL,批内及批间变异系数均低于3 %,对人工污染的脱脂牛奶、LB液体培养基中肠毒素蛋白SEP的回收率高于98%。结论 本实验建立了一种能够快速检测葡萄球菌肠毒素SEP的ELISA方法。     

检测赭曲霉毒素A(OTA)的酶联免疫吸附法(ELISA)体系的建立

本文分别以自制抗-OTA兔血清抗体(IgG)及鸡卵黄抗体(IgY)就影响检测OTA的ELISA(酶联免疫吸附法)体系因素进行了探讨,建立了检测OTA的ELISA方法。鸡卵黄抗体的最佳ELISA操作参数为:包被抗原浓度为7.5μg/ml,封阻剂浓度为2.5%,抗体稀释倍数为1000,酶标二抗稀释倍数为14000;兔血清抗体的最佳ELISA操作参数为:包被抗原浓度为2.5μg/ml,封阻剂浓度为2.5%,抗体稀释倍数为8000,酶标二抗稀释倍数为10000。IgG和IgY的检测灵敏度分别为10ng/ml和1ng/ml。     

多重PCR检测金黄色葡萄球菌六型肠毒素基因的研究

目的:为了建立一种简单、快速检测金黄色葡萄球菌六型肠毒素基因的多重PCR方法。方法:根据相关文献和Genebank报道的编码金黄色葡萄球菌肠毒素A、B、C、D、E、H的基因序列,选择合成了6对特异性引物,建立多重PCR体系,并对反应条件进行了优化。结果:6对引物能同时特异地扩增出120、478、257、319、170、375bp的目的片段,表明6对引物具有良好的特异性。结论:成功地建立了一种同时检测金黄色葡萄球菌六型肠毒素基因的多重PCR方法,在金黄色葡萄球菌肠毒素快速筛查方面具有良好的应用前景。     

ELISA法快速检测水产品中副溶血性弧菌

以副熔血性弧菌抗原免疫新西兰大白兔获得特异性抗体,利用此抗体,进行间接竞争ELISA法实验,确定了抗原抗体最佳反应浓度和一抗稀释度分别为107cfu/mL和1:4000,酶标二抗IgG-HRP最佳工作稀释度为1:1000.以此方法分别对人工染菌水产品及实际水产品进行检测,检测下限为104cfu/mL,检测时间为8h;而经8h增菌后,其检测下限为103cfu/mL.此法与常规方法检测结果完全一致,具有很高的实际应用价值.     

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.     

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     

Chinese CTP Market

According to Printing and Printing Equipment Industries Association of China（PEIAC）＇s statistics to the plate manufucturer in China, in 2013, the actual offset plate production has reached 346 million square meters in China. Among them, the CTP production volume was 245 million square meters, up by 11% than that of last year; the total sales of the CTP plate was 239 million square meters, up by 13%.     

Preparatory Work of Print China 2015 is Going Smoothly

正Held at Guangdong Modern International Exhibition Center,Print China 2015 will cover 7exhibition halls,besides the original Hall No.3,4,5,6,7,the newly built F zone of Hall 3 will be used too.The total area will be140,000 square meters.Hall 3:Offset and large printing equipment,package printing equipment,post press