食用海藻种属特异性多重PCR鉴定

为了对食用海藻进行种属特异性分子特征鉴定,采用试剂盒提取4种常见的食用海藻(石莼、龙须菜、海带、螺旋藻)样品DNA后,用常规PCR技术进行引物特异性验证,运用双重PCR技术检测引物的灵敏度。结果表明,4种食用海藻(石莼、龙须菜、海带、螺旋藻)得到相应的不同大小单一条带,说明引物具有特异性;在双重PCR体系螺旋藻、石莼以及龙须菜、海带组合,得到石莼的检测限为362 pg,龙须菜的检测限为100 pg。     

麦芽品种DNA指纹图谱构建的研究

麦芽作为啤酒的主要原料,麦芽品种折鉴定对啤酒酿造至关重要。微卫星DNA(SSR)作为第二代分子标记技术,在植物物种多样性和品种鉴定上应用广泛,因此本研究利用SSR分子标记进行麦芽品种DNA指纹图谱构建。结果表明,从105对SSR引物中筛选到6对多态性丰富的SSR引物,每对引物的等位基因3~10个不等,平均每个引物产生5.83个等位基因。对实验样品的等位基因进一步分析发现,仅需4对核心引物就可以实现21个麦芽品种的区分。利用该技术,我们建立了相应麦芽品种DNA指纹图谱,为啤酒企业的麦芽采购提供技术支持。     

烟草拟茎点霉快速检测方法的建立

为实现烟草拟茎点霉的快速准确检测,利用真菌rDNA-ITS通用引物对拟茎点霉和其他10种烟草病原菌基因组DNA进行了PCR扩增、片段回收及DNA测序,在序列比对基础上设计了两对特异性检测引物,并鉴定了引物的检测灵敏度和特异性,同时优化建立了PCR反应体系。结果表明,在退火温度为60 ℃时,两对特异性引物P2F/P3R和P3F/P3R从烟草拟茎点霉DNA样本中分别扩增出来420 bp和381 bp的特异性条带,而在其他10种烟草病原菌DNA以及阴性对照中均不能扩增到任何条带。两对特异性引物的检测灵敏度均达到10 pg/μL。建立的基于两对特异性引物P2F/P3R和P3F/P3R的分子检测方法可实现对病原菌烟草拟茎点霉的快速准确检测。      

利用DNA分子指纹图谱技术检测小麦粉中害虫的研究

为开发快速检测我国成品粮中害虫的技术,研究了DNA分子指纹图谱技术检测小麦粉中主要害虫的碎片和虫卵的含量。结果表明,通过对害虫的延长因子EF1基因片段引物的筛选和PCR条件的选择,确定了有效地检测出来小麦粉中的赤拟谷盗、杂拟谷盗和锯谷盗的成虫碎片、幼虫和卵通用引物和PCR扩增条件。通过改良的PCR技术可在4 h内快速检测杂拟谷盗、锯谷盗的卵、幼虫和成虫碎片。最低检出限为可检测出小麦粉中0. 05%(m/m)卵和幼虫、1%成虫的碎片,灵敏度超过了国际标准。DNA指纹图谱中电泳条带亮度(Y)与被检测的不同害虫碎片含量(X)成正相关,回归方程为Y=858. 36X+74. 540相关系数R为0.961 9。利用该技术检测随机取样的小麦粉中成虫的碎片,检测的准确率最高可达80. 56%。结果说明了利用DNA分子指纹图谱技术可快速检测出小麦粉中害虫的碎片、幼虫和卵含量,并可预测小麦粉中害虫的感染程度。本研究为开发快速小麦粉中害虫的快速检测技术具有重要的参考意义。     

4组鱼类DNA条形码引物的筛选与优化

目的通过对DNA条形码氧化酶亚基I基因(cytochrome oxidase subunit I,COI)的4组常用引物进行比较筛选,选择适于舟山鱼类鉴定的最佳引物。方法以舟山主产大黄鱼、小黄鱼、带鱼和乌贼样品DNA为模板,对4组常用DNA条形码引物进行PCR体系优化。通过比较PCR产物量、特异性、灵敏度和测序成功率,综合分析筛选最佳引物组。结果 4组引物均能扩增大黄鱼、小黄鱼和带鱼的DNA,对于乌贼DNA的扩增具有明显差异。COI优化引物具有易于扩增、测序简单等优势,更适于作为海产品鉴定的DNA条形码引物。结论本研究为舟山海产品的市场监管和实验室大量样品检测提供了技术参考。     

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.     

基于锁核苷酸(LNA)增敏的植烟土壤中烟草黑胫病菌定量PCR检测方法

为了快速有效地检测植烟土壤中的烟草黑胫病菌的定殖数量,以一种锁核苷酸(LNA)引物为基础,进行了植烟土壤中烟草黑胫病菌数量的定量PCR 检测方法研究。结果表明:①与普通DNA引物相比,LNA 引物提高了PCR 反应的退火温度,减少了引物二聚体和非特异性产物的形成,提高了烟草黑胫病菌分子检测的灵敏度与特异性;②定量分析检测出14 个烟草-大蒜轮作土样中烟草黑胫病菌的定殖数量为2.76×103～5.20×104个/g,且在4～5 h 内完成检测,具有较高的灵敏度和检测效率。      

公共引物介导的多重定量PCR技术在转基因大豆检测中的应用研究

目的 建立公共引物介导的多重定量PCR法检测我国农业农村部允许进口的6种转基因大豆(包括: MON87701、DP356043、CV-127、MON87769、MON87705、MON87708)的方法。方法 设计并筛选出针对本研究涉及的6种转基因大豆对特异性引物, 在每对特异性引物的5’端均加上一段公共引物, 形成特异性嵌合引物。以某种转基因大豆DNA为模板, 加入多引物体系以及荧光公共引物验证GeXP(gene expression platform)多重检测体系的特异性, 每种转基因大豆DNA模板浓度分别稀释为20、2、1、0.2、0.02 ng/μL, 运用GeXP多重PCR方法进行单一DNA模板灵敏度分析并优化各对特异性嵌合引物的工作浓度。结果 多重PCR检测体系扩增出特异性片段, GeXP检出的各转基因大豆片段长度MON87701、DP356043、CV-127、MON87769、MON87705和MON87708分别为140.75、184.42、274.93、308.47、467.98和517.67 bp, GeXP 多重检测体系检测敏感性可达到为1 ng/μL, 扩增产物测序结果进一步说明了引物扩增的特异性。结论 本研究建立的基于GeXP系统的多重PCR定量检测技术, 可以准确鉴别6种转基因大豆, 为转基因大豆及其他转基因产品提供了新型的检测方法。     

3种血清型沙门氏菌多重PCR检测方法的建立

目的建立多重聚合酶链式反应(multiplex polymerase chain reaction,mPCR)法检测3种常见血清型沙门氏菌的分析方法。方法以肠炎沙门氏菌Hat基因、鼠伤寒沙门氏菌Stm-4495基因、乙型副伤寒沙门氏菌sdfI基因设计合成特异性引物,提取沙门氏菌基因组DNA为扩增模板,验证引物特异性,优化多重PCR退火温度及引物浓度,检测方法特异性及检出限,并利用该方法对人工污染冷鲜鸡肉进行检测。结果 3对引物特异性强且无交叉影响;25μL多重反应体系中,引物STM、HAT、SDF最佳终浓度分别为:0.5、0.5、0.4μmol/L,最佳退火温度为60℃;11株阴性对照菌无目标条带检出,方法特异性良好,以3种血清型沙门氏菌纯培养物DNA混合物为模板,检出限低至DNA质量浓度1 pg/μL;人工污染鸡肉经过12 h增菌,能同时检测出3种血清型沙门氏菌的检测限为:肠炎沙门氏菌(4.0±1.0) CFU/g、鼠伤寒沙门氏菌(8.0±0.9) CFU/g、乙型副伤寒沙门氏菌(8.0±0.6) CFU/g。结论本方法特异性强、检测限低,对食品中3种常见血清型沙门氏菌快速检测具有重要意义。     

基于线粒体DNACOⅠ基因鉴别畜禽肉中鸡源性成分

为建立畜禽肉中鸡源性成分的快速检测方法,以线粒体DNA细胞色素C氧化酶亚基Ⅰ(COⅠ)基因序列为靶点,比较羊、牛、猪、兔、鸽、鹌鹑、鸡、鸭、鹅9种动物COⅠ基因的差异位点,设计筛选鸡特异性引物,进行常规聚合酶链式反应(polymerase chain reaction,PCR)和荧光定量PCR检测。结果表明:所设计的引物仅对鸡肉DNA模板有扩增条带和典型扩增曲线,且循环阈(cycle threshold,Ct)值为21.78,对其他动物DNA模板无扩增。方法特异性较强,灵敏度较高,达pg级,可以用于畜禽肉与肉制品中鸡源性成分的快速、有效、准确检测。     

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.     

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.     

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.     

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.     

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.     

Analysis on Economic Indicators from 62 Enterprises in Printing Machinery Industry in Q1 2014

Operation of printing machine industry was still unsatisfactory in the first quarter of 2014.Analysis on operation of printing machine industry.a.Market demand was not strong;sales of product undulated and declined.According to the statistics,the total industrial output value fell by 19.28% in the first quarter of 2014 than the average quarter value in 2013; industrial added value decreased by 4.16%; sales revenue dropped by 22.83%. h. Business operation of enterprises was in poor condition. c. R＆D of new products is an important transformation guarantee for enterprises. d. To take self explore new ways upgrading advantages,and explore new ways.