食品中单核细胞增生李斯特菌微滴数字PCR定量检测方法建立

目的 建立食品中单核细胞增生李斯特氏菌的微滴式数字聚合酶链式反应（ddPCR）快速定量检测方法。方法 筛选单核细胞增生李斯特氏菌的特异性引物和探针,通过对目标菌纯菌液及人工污染样品的检测,比较ddPCR方法和平板计数法的定值效果,对ddPCR结果进行特异性、灵敏性和重复性分析。结果 本研究建立的单核细胞增生李斯特氏菌ddPCR检测方法具有良好的特异性、灵敏性和重复性。单核细胞增生李斯特氏菌纯菌液中定量限（LOQ）和检出限（LOD）均为136 CFU/mL,在鱿鱼圈和香肠样品中定量限分别为240 CFU/g和155 CFU/g。ddPCR在各梯度水平上变异系数均小于25%,ddPCR和平板计数定值对数值相对偏差均小于30%。结论 本研究建立的ddPCR方法能够快速、准确、灵敏、特异地定量检测食品中单核细胞增生李斯特氏菌。     

微滴数字PCR定量检测乳制品中单核细胞增生李斯特氏菌

选取单核细胞增生李斯特氏菌iap基因为靶基因,利用微滴数字PCR(ddPCR)技术对乳制品中单核细胞增生李斯特氏菌进行定量检测。实验结果表明,本方法能够对含菌量在5×10~3～5×10~6CFU/mL之间的巴氏乳样品进行准确定量检测,检测下限和定量检测下限分别为5×10~2CFU/mL和5×10~3CFU/mL,检测的准确性和灵敏性均优于qPCR检测。     

PMA与ddPCR结合检测单核细胞增生李斯特氏菌

将叠氮溴化丙锭(PMA)与微滴数字PCR(dd PCR)技术相结合,检测热灭活背景下单核细胞增生李斯特氏菌活菌。结果表明,PMA终浓度为5.0μg/m L、曝光时间为15 min时,可以有效抑制10~5 CFU/m L的单核细胞增生李斯特氏菌死菌DNA的PCR扩增,不抑制单核细胞增生李斯特氏菌活菌DNA扩增的PMA最高浓度是10.0μg/m L。利用PMA处理死活菌混合液时,PMA-dd PCR可以在死菌存在的条件下,定量检测活菌,降低了"假阳性"结果的出现。检测结果显示:PMA-dd PCR灵敏度是2.0 copy/20μL。利用PMA-dd PCR检测人工污染的鳕鱼样品,最低可检出10~2 CFU/m L的单核细胞增生李斯特氏菌。结果证明PMA-dd PCR方法的精确度、稳定性良好。     

水产品中创伤弧菌ddPCR定量方法的建立

选取创伤弧菌单拷贝基因met为靶基因,设计引物探针,建立对创伤弧菌准确定量的微滴式数字聚合酶链式反应(droplet digital polymerase chain reaction,ddPCR)方法,并进行特异性、灵敏度和重复性实验,同时与实时PCR(real-time PCR)方法进行比较。结果显示,所建立的ddPCR方法可以快速、高效地检测出创伤弧菌,细菌纯培养物中其定量限可达323拷贝数/mL,检测限可达61拷贝数/mL,人工污染牡蛎样品中能最低能检测到1.13×102拷贝数/g的目标菌。对人工污染样品中目标菌的检测,ddPCR的定量结果约为平板计数结果的1.4倍,比real-time PCR方法的检测更加稳定准确。本研究建立的ddPCR检测方法能快速准确、特异、灵敏地定量检测创伤弧菌。     

单核细胞增生李斯特菌荧光定量PCR检测试剂盒的研制

目的:建立一种用于单核细胞增生李斯特菌快速检测的荧光定量PCR试剂盒.方法:根据本课题组前期工作基础,针对单核细胞增生李斯特菌hly基因序列设计TaqMan探针和引物,建立荧光定量PCR检测方法,将反应试剂组装成试剂盒,并对该试剂盒的各项参数进行评价.结果:特异性试验表明,组装的试剂盒对8林单核细胞增生李斯特菌标准菌株的检测均呈阳性反应,对6株其它种的李斯特菌及13株食品(环境)中常见的非李斯特污染菌(如沙门氏菌、金黄色葡萄球菌)呈阴性反应.该试剂盒的基因组DNA检测灵敏度为3.94fg/反应(约合1.34CFU/反应).该试剂盒批内平均变异系数为2.2%,批间变异系数为1.9%,具有良好的重复性;同时稳定性强,反复冻融处理对其无明显影响;可在-20℃下避光保存1年.结论:该试剂盒具有特异性强、灵敏度高、重复性好、稳定性强等优点,可应用于单核细胞增生李斯特菌的快速检测.     

三重微滴数字PCR定量检测即食米粉中致病菌方法研究

目的 建立三重微滴数字PCR（ddPCR）方法同时定量检测即食食品中的沙门菌、蜡样芽胞杆菌和单核细胞增生李斯特菌。方法 选择以沙门菌ttrA/ttrC、蜡样芽胞杆菌essC、单核细胞增生李斯特菌侵袭相关内肽酶基因等3个单拷贝基因对应的3对引物探针，采用实时荧光定量PCR验证引物/探针特异性后，建立三重ddPCR方法同时定量检测3种致病菌的拷贝数。结果 该方法的线性范围分别为：沙门菌25~22 687 copies/20 μL；蜡样芽胞杆菌19~15 620 copies/20 μL；单核细胞增生李斯特菌18~23 373 copies/20 μL，线性相关因子r²≥0.999，6个浓度3次重复测定3种菌的相对标准偏差（RSD）≤12%，重复性好，对于上述菌株的最低检出限分别为6、3和7 copies/20 μL；采用已建立的ddPCR方法和平板计数方法对模拟染菌米粉样品进行检测，两种方法测定值结果RSD小于9%，结果一致性较好。结论 本研究建立的三重ddPCR同时定量检测即食食品中沙门菌、蜡样芽胞杆菌和单核细胞增生李斯特菌的方法与平板计数法相比，更快速、灵敏，结果准确。     

基于双启动引物特异性检测单核细胞增生李斯特氏菌的PCR方法

以单核细胞增生李斯特氏菌iap基因为靶基因,利用一新型PCR引物设计方法--双启动引物(Dual-priming oligonucleotide,DPO),建立了特异性检测单核细胞增生李斯特氏菌的DPO-PCR方法,测试了DPO-PCR方法退火温度不敏感性、特异性及灵敏度,并在实践检测中进行了初步应用。结果显示:该方法检测单核细胞增生李斯特氏菌的灵敏度为1.51×102CFU/mL;退火温度不敏感性测试中,与常规PCR引物相比,DPO引物在48~68℃退火温度范围内均能够高效率地扩增靶基因;特异性测试中,DPO-PCR方法能特异地检测出目标菌,与其他菌株无非特异性扩增反应,比常规PCR方法显示出更强的特异性。实践应用证明,利用DPO-PCR方法对130份样本进行检测,共计检出9份单核细胞增生李斯特氏菌阳性样本,经国标法(GB/T 4789.30-2008)复检,两者检测结果一致,显示出良好的实用性,为单核细胞增生李斯特氏菌的快速准确检测提供了新方法。     

双重荧光定量PCR检测沙门菌和单核细胞增生李斯特菌方法的建立

目的建立双重荧光定量PCR方法,快速检测沙门菌和单核细胞增生李斯特菌。方法通过设计特异性引物和探针,扩增沙门菌的fimY基因和单核细胞增生李斯特菌的hly基因,采用倍比梯度稀释法检测该体系的灵敏度,以另外7株肠道致病菌评价检测体系的特异性;建立了沙门菌和单核细胞增生李斯特菌感染小鼠的检测模型以验证方法的适用性。结果建立了同时检测沙门菌和单核细胞增生李斯特菌的双重荧光定量PCR方法,从DNA提取到检测完毕仅需2.5 h。检测两种病原菌的灵敏度分别为11和12.8 copies/μl,特异性为100%,符合率为93.3%。结论该法缩短了检测时间,并有良好的灵敏性和特异性,在疾病防控及食品卫生行业中很有应用前景。     

FTA滤膜用于PCR检测单核细胞增生李斯特氏菌的研究

建立单核细胞增生李斯特氏菌(Listeria moncytones,LM)快速、敏感、特异的PCR检测方法.利用FTA滤膜提取模板DNA,采用PCR特异性扩增单增李斯特菌的溶血素基因(HIyA),并评价该方法的特异性与灵敏性.引物能特异性的扩增单增李斯特的HIyA基因,而其他细菌的扩增结果均呈现阴性:利用FTA滤膜提取模板直接检测单增李斯特具有较高的灵敏度,灵敏度为l 02 cfu/mL.利用FFA滤膜提取模板,操作简便,成本低且具有较高的灵敏度,为食品中单核细胞增生李斯特氏菌的快速检测提供新的手段.     

应用基因芯片技术检测肉及肉制品中5种致病菌

建立一种运用多重聚合酶链式反应(PCR)结合基因芯片技术检测大肠埃希氏菌、沙门氏菌、金黄色葡萄球菌、志贺氏菌和单核细胞增生李斯特菌5种食源性致病菌的快速、准确、灵敏的方法。分别选取编码大肠埃希氏菌的slt基因、沙门氏菌invA基因、金黄色葡萄球菌nuc基因、志贺氏菌ipaH基因和单核细胞增生李斯特菌inlA基因,并以细菌16S rDNA基因作为阳性对照,设计引物和探针,进行多重PCR扩增,产物与含特异性探针的芯片杂交。结果表明：该基因芯片可同时特异性地检测5种致病菌,多重PCR检测灵敏度为20pg,而DNA芯片检测灵敏度可达2pg;用所制备的基因芯片检测实际肉及肉制品样品,准确率高于传统培养法。所建立的基因芯片检测方法特异性好、灵敏度高,可为食源性致病菌的检测提供理想手段。     

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.     

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.     

Review of techniques to manufacture micro-hydrogel particles for the food industry and their applications

Microgels are ‘soft’ microscopic cross-linked polymeric particles that are being increasingly exploited in a variety of industries for rheology control, encapsulation and targeted delivery. They are valued because of the ability to tune their functionality to address specific applications in oil recovery, coatings, drug delivery, cosmetics, personal care and foods. Food microgels are typically biopolymer hydrogels in the form of microspheres, nanospheres (also called nanogels), spheroids and fibres. The utilisation of engineered microgels in foods has so far been limited, despite their great potential to address several needs in the food industry, including: satiety control, encapsulation of phytonutrients and prebiotics, texture control for healthier food formulations (e.g. reduced fat products), and targeting delivery to specific areas in the digestive tract. We review the scientific and patent literature on the utilisation and manufacturing methods for producing microgels with an emphasis on micro-hydrogels for food applications.     

12—A CRITIQUE OF SOME HYPOTHESES RELATING TO THE HETEROGENEITY OF THE HIGH-SULPHUR PROTEINS OF WOOL

Joubert and Burns prepared a large number of fractions from the high-sulphur proteins of wool and estimated their molecular weights and amino-acid compositions. Their data have been re-examined in order to look for statistically significant interrelations between amino acids and between the proportion of various amino acids and molecular weight. Statistical analysis of the data is also used to examine the credibility of some hypotheses concerning the mechanism of keratin biosynthesis and to provide further evidence for the existence of families of proteins within the high-sulphur fractions of wool.     

Contribution of European research to risk analysis

The European Commission's, Quality of Life Research Programme, Key Action 1—Health, Food & Nutrition is mission-oriented and aims, amongst other things, at providing a healthy, safe and high-quality food supply leading to reinforced consumer confidence in the safety of European food. Its objectives also include the enhancing of the competitiveness of the European food supply. Key Action 1 is currently supporting a number of different types of European collaborative projects in the area of risk analysis. The objectives of these projects range from the development and validation of prevention strategies including the reduction of consumers risks; development and validation of new modelling approaches; harmonization of risk assessment principles, methodologies, and terminology; standardization of methods and systems used for the safety evaluation of transgenic food; providing of tools for the evaluation of human viral contamination of shellfish and quality control; new methodologies for assessing the potential of unintended effects of genetically modified (genetically modified) foods; development of a risk assessment model for Cryptosporidium parvum related to the food and water industries; to the development of a communication platform for genetically modified organism, producers, retailers, regulatory authorities and consumer groups to improve safety assessment procedures, risk management strategies and risk communication; development and validation of new methods for safety testing of transgenic food; evaluation of the safety and efficacy of iron supplementation in pregnant women; evaluation of the potential cancer-preventing activity of pro- and pre-biotic ('synbiotic') combinations in human volunteers. An overview of these projects is presented here.     

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 Latest Data about Development of Chinese Printing Industry in 2013

In the 2014 China（Shanghai）International Printing Week,Director Wang Yanbin released the latest data about development of Chinese printing industry in 2013.According to statistics,in 2013,the total output value of Chinese printing industry exceeded 1trillion Yuan for the first time,reaching 1.03985 trillion Yuan.There were 105,000 printing enterprises in China,employees were 3.415 million.The total asset was 1.06247 trillion Yuan;     

The State Council Issued "Directory of Government Approved Investment Projects(2013 Edition)"

正On December 2nd,2013,the State Council issued the notification of"Directory of Government Approved Investment Projects(2013 Edition)"(hereafter referred to as"notification").It is pointed out in the"notification"that in order to further deepen reforms in investment systems and administrative examination and approval systems,simplify administrative procedures and delegate powers to lower levels,earnestly     

China textile machinery: taking off in progress

正Among the 1600 exhibitors who take apart in the ITMA ASIA+CITME2014 2/3 are Chinese manufactures.If the numerous figures failed to attract your attention,the increase of quality should draw your focus.To adopt the demand of developing textile machine market,domestic textile machinery enterprises now follow the slogan of"technology drives development"to enhance product competitiveness.Our domestic sellers will showcase product ranging from spinning,weaving,dyeing and printing,     

Top Ten News of China's Paper Industry 2013

On December 24th, 2013, the meeting on the selection of top 10 news of China＇s paper industry 2013 sponsored by 〈China Paper Newsletters〉 was held in Beijing. The yearly selection of the top l0 news, which began in 2000, has become a brand activity widely recognized in the industry thanks to the support from the authorities at all levels and public participation.