产单核李斯特菌的改良分子信标荧光PCR-磁捕获快速检测体系的建立

[目的]建立改良分子信标荧光PCR-磁捕获快速检测产单核李斯特菌(LMO)的体系,应用于食品中LMO的污染状况调查及食物中毒快速诊断. [方法]根据GenBank公布的LMO hlyA基因的保守序列,设计引物和改良分子信标探针,建立改良分子信标荧光PCR检测体系.运用磁捕获材料对PCR检测阳性的增菌液进一步处理后划平皿做分离培养. [结果]改良分子信标-实时PCR反应体系DNA灵敏度为110 fg,菌液灵敏度为99 cfu/ml或4 cfu/PCR反应体系,无交叉反应.以此反应体系检测28株LMO,均出现特异的荧光信号.对228份食品进行LMO检测,8份增菌液LMO实时荧光PCR阳性.用磁捕获材料处理此8份增菌液,全部细菌培养阳性,高于国标法的6份阳性.[结论]改良分子信标一实时PCR反应体系快速、灵敏度高,特异性强,运用磁捕获试剂能有效提高培养阳性率.应用该检测系统能提高LMO的检出率和准确性.该系统可应用于LMO食品污染状况调查及食物中毒的快速诊断.     

改良分子信标-实时PCR快速检测产单核李斯特菌

目的：建立改良分子信标-实时PCR检测产单核李斯特菌（LMO）的快速方法，应用于食品中LMO的污染状况调查及食物中毒快速诊断。方法：根据GenBank公布的LMO hlyA基因的保守序列，设计引物和改良分子信标探针，建立改良分子信标-实时PCR检测体系，应用于食品中LMO检测。结果：改良分子信标-实时PCR反应体系DNA灵敏度为110fg，菌液灵敏度为99cfu／ml或4cfu／PCR反应体系，无交叉反应。以此反应体系检测28株LMO，均出现特异的荧光信号。上述方法可将检测时问由原来的至少4d缩短至1d。对228份食品进行LMO检测，8份增菌液LMO实时荧光PCR阳性，其中6份1310细菌培养阳性。结论：改良分子信标-实时PCR反应体系快速、灵敏度高，特异性强．能提高LMO的检出率和准确性，可应用于LMO食品污染状况调查及食物中毒的快速诊断。     

改良分子信标—实时PCR快速检测副溶血弧菌

目的 :建立改良分子信标 -实时 PCR检测副溶血弧菌的快速方法 ,应用于副溶血弧菌食物中毒的快速诊断和海产品检验。方法 :根据 Gen Bank公布副溶血弧菌的耐热直接溶血毒素基因 (TDH)的保守序列 ,设计一对引物和改良分子信标探针 ,用 FAM荧光剂标记探针的 5′,并进行特异性和灵敏度分析 ;同时以 11种细菌作对照 ,建立改良分子信标检测副溶血弧菌的实时 PCR反应体系 ,应用于副溶血弧菌食物中毒快速诊断和食品微生物检测。结果 :检测 12种细菌 ,只有副溶血弧菌有荧光信号 ,与其他细菌无交叉反应 ,DNA灵敏度为 16 6 .6 fg/ μl,菌液灵敏度为 6 9cfu/ ml或 6 cfu/ PCR反应体系。改良分子信标 -实时 PCR反应体系检测 4 0株副溶血弧菌均出现特异的荧光信号 ,无干扰。对 3起细菌性食物中毒共 4 8份样品和 10 0份海产品进行检测 ,9份副溶血弧菌实时 PCR阳性 ,其中 7份副溶血弧菌细菌培养阳性 ,其余样品都为阴性。检测时间仅需 2 h。结论 :改良分子信标 -实时 PCR检测体系快速、灵敏度高 ,特异性强 ,可用于副溶血弧菌食物中毒的快速诊断 ,为食源性疾病的分子流行病学调查提供新的检测手段     

改良分子信标-实时PCR快速检测志贺菌

目的：建立改良分子信标-实时PCR检测志贺菌的快速方法，应用于志贺菌食物中毒的快速诊断和门诊肠道致病菌的检测。方法：根据GenBank公布志贺菌ipaH基因的保守序列，设计引物和改良分子信标探针，建立实时PCR-改良分子信标检测体系，应用于对志贺菌食物中毒的快速诊断和门诊肠道致病菌的检测。结果：改良分子信标-实时PCR反应体系DNA灵敏度为93fg/μl，菌液灵敏度为64cfu／ml或2cfu／PCR反应体系，无交叉反应。此反应体系检测67株志贺菌，均出现特异的荧光信号。对细菌性食物中毒样本等共657份样品进行志贺菌检测，42份志贺菌实时PCR阳性，其中41份志贺菌细菌培养阳性。从样品处理到检测结果仅需2h～1d时间。结论：改良分子信标-实时PCR检测体系快速、灵敏度高，特异性强，可用于志贺菌食物中毒的快速诊断，为食源性疾病的分子流行病学调查提供新的检测手段，对于提高肠道门诊的工作效率有重要意义。     

双重实时荧光PCR快速检测O157大肠杆菌的初步研究

[目的]建立双重实时PCR体系,实现对O157大肠杆菌rfbE和stx2基因的同步检测。[方法]根据GenBank公布的O157大肠杆菌rfbE和stx2基因序列,应用分子生物学软件设计两对引物和TaqMan探针,并对荧光定量PCR反应条件进行优化,建立实时荧光定量PCR检测O157大肠杆菌的反应体系,并对该法的特异性、敏感性和重复性进行了评价。[结果]所有O157大肠杆菌菌株的检测结果均为阳性,而所有其他菌株检测结果均为阴性;该方法对O157大肠杆菌纯培养的检测范围为10^0～10^6cfu/μl,重复性检测的变异系数均小于5%。对模拟污染牛奶样本的检测范围为10^2～10^6cfu/μl。从细菌核酸提取至完成检测约需3h。[结论]基于Taqman探针技术的实时PCR检测体系可同步检测O157大肠杆菌rfbE和stx2基因,具有快速、灵敏度高、特异性强等优点,可用于O157大肠杆菌食物中毒的快速诊断和食品微生物检测。     

大肠埃希菌O157:H7的实时荧光PCR检测方法研究

目的建立一种敏感、特异、快速的大肠埃希菌O157:H7的检测方法,应用于突发公共卫生事件及食源性致病菌流行病学调查的检测。方法根据GenBank大肠埃希菌O157:H7rfbE基因序列,设计引物和TaqMan探针,对实时荧光PCR反应条件进行优化,建立实时荧光PCR检测大肠埃希菌O157:H7的反应体系,并对该法的特异性、敏感性和重复性进行评价。结果大肠埃希菌O157:H7菌株的检测结果均为阳性,而所有其它菌株检测结果均为阴性;该方法检测的灵敏度可达1×102cfu/ml。模拟污染的猪肉、羊肉、鸡肉、生食蔬菜样品,均可检出1×104cfu/ml的细菌。从细菌核酸提取至完成检测约需3 h。结论建立的实时荧光PCR检测方法具有灵敏度高、特异性强、快速等优点,可用于大肠埃希菌O157:H7食物中毒的快速诊断和食品微生物检测,为食源性疾病的分子流行病学调查提供新的检测手段。     

荧光PCR检测副溶血性弧菌

[目的]建立荧光PCR检测副溶血性弧菌的快速方法。[方法]根据基因库公布的副溶血性弧菌的耐热直接溶血素基因（TDH）的保守序列,设计一对引物,用FAM荧光剂标记探针的5＇,进行特异性和灵敏性分析,使用于食物中毒样品的快速检测。[结果]检测灵敏度为170.8fg/μl,菌液的灵敏度为30~70cfu/ml。用此反应体系检测53株副溶血性弧菌均出现特异的荧光信号,未见与其它试验种属细菌交叉。对3起食物中毒样品45份用荧光PCR法和国标法检测副溶血性弧菌,荧光PCR检测阳性22份,国标法阳性20份。荧光PCR检测从样品处理到结果只需8h。[结论]荧光PCR检测副溶血性弧菌灵敏度高、特异性强、简便快速,可用于副溶血性弧菌引起食物中毒的快速诊断和海产品副溶血性弧菌的快速检测。     

副溶血性弧菌所致食物中毒实时荧光定量PCR筛查方法的建立及应用

目的建立副溶血性弧菌(VP)的实时荧光定量PCR测定方法。方法利用副溶血性弧菌toxR基因DNA序列设计引物和探针,用细菌标准菌株检测方法的特异性,VP纯培养液及模拟污染食物测定方法的灵敏度和重复性,并应用在2012年-2014年疑似细菌食物中毒调查中。结果当标准曲线在2.5×101copy~2.5×106copy时,线性关系良好,相关系数为0.998 6,线性方程为y=-3.413 2x+38.919 5;仅VP标准菌出现典型的"S"形荧光扩增曲线;质粒检测灵敏度为25 copy/ml,直接能检测到的最低菌液浓度:纯培养液为21 cfu/ml,模拟食品为1.8×102cfu/ml;同一模拟食品(1.8×104cfu/ml)测定的重复性好(批内Ct值变异系数为1.12%,批间变异系数为1.70%);食物中毒RT-PCR结果为阳性的26份肛拭中16份分离到VP,1份阳性食品分离出VP,未发现假阳性。结论本法快速、敏感、特异性好,在细菌性食物中毒检测的筛查中可大大提高检测效率。     

TaqManTM实时荧光PCR快速检测沙门氏菌的初步研究

[目的]建立实时荧光TaqManTM检测沙门氏菌的快速检测方法,为食物中毒快速准确的定性检测及食品中沙门氏菌染菌的调查提供手段,也为建立实时荧光TaqManTM同时检测多种细菌性食物中毒目标菌奠定基础. [方法]通过分析沙门氏菌invA、hilA、fimA、hns、spy、hut基因和16S rDNA等目的基因,对比不同目的基因的优缺点,最终选择hut基因作为目的基因,在Genebank进行中查找多条hut基因序列,并利用DNAssist软件进行同源性分析,选取相对保守区段,用Beacon Designer软件自行设计引物和TaqManTM探针,并利用硅胶模型基因组DNA提取法制备基因组DNA,通过对TaqManTM实时荧光PCR缓冲液浓度、Mg2+浓度、dNTP浓度、Taq酶浓度、引物浓度和探针浓度等反应条件的优化,初步建立沙门氏菌的TaqManTM实时荧光PCR快速检测方法. [结果]选取的hut基因同源性达到99％以上,特异性好.通过对各实验条件的优化,得到沙门氏菌TaqManTM实时荧光PCR的最佳反应条件,初步建立TaqManTM实时荧光PCR检测沙门氏菌的快速检验方法,该方法能在1h内完成整个检测过程.[结论]TaqManTM实时荧光PCR检测不但灵敏度高、特异性好,而且检测周期短,能提高沙门氏菌的检出率和检测准确性,可望应用于沙门氏菌食品及食物中毒的快速定性检测.     

食品中产气荚膜梭菌α毒素基因快速检测方法的研究

目的建立分子信标荧光PCR体系检测产气荚膜梭菌的快速检测方法,应用于食物中毒快速诊断和食品微生物检验。方法根据GenBank公布的保守序列,针对α毒素基因设计一对引物和分子信标探针,用FAM荧光剂标记探针的5’端,并进行特异性和灵敏度分析,同时以10种细菌作对照。结果分子信标荧光PCR反应体系检测10种细菌,只有产气荚膜梭菌出现特异荧光信号,其他均无荧光信号,而且与其他细菌无交叉反应。对40份食品样品进行检测,3份产气荚膜梭菌PCR阳性,其余样品为阴性,检测仅需2 h。3份阳性样本经传统方法培养,2份有检出产气荚膜梭菌。结论分子信标荧光PCR检测体系快速、灵敏度高,特异性强,可用于产气荚膜梭菌食物中毒的快速诊断和食品污染物及感染性腹泻等监测工作中,为食源性疾病的分子流行病学调查提供新的检测手段。     

Calcium intake in the elderly: validation of a dietary questionnaire

Epidemiological studies of calcium and osteoporosis have been hampered by the lack of a suitable tool for assessing calcium intake. This report describes a new frequency and amount questionnaire for measuring present and past calcium intake in the elderly. The validity of the questionnaire was tested against two commonly used standards of dietary assessment, five-day duplicate diets and seven-day weighed dietary inventories. The resulting correlation coefficients were, respectively, r = 0.76 and r = 0.69, while that for repeatability was r = 0.84. Furthermore, the questionnaire categorized subjects into thirds of the distribution of intake with almost no gross misclassification. It is suggested that the present findings may be extended to the majority of normal, healthy elderly subjects, implying wide application for the questionnaire in the assessment of calcium intake in the elderly.     

Self-reported health problems among Swedish miners one year after unemployment

Unemployment is considered to be a public health concern sincedeterioration in the health of the unemployed is often anticipated.However, for some groups, such as miners, unemployment mightimprove health due to a cessation of potentially harmful occupationalexposures. This study evaluates the health of 79 miners in oneSwedish iron-ore mine, and 226 age-matched controls from thegeneral population, during one year after the closure of themine. The participants received a questionnaire regarding medicalhistory and subjective symptoms at the beginning of the studyperiod, and after one year. Statistically significant negativeeffects on self-reported health attributable to unemploymentwere not found, although neuropsychiatric symptoms were morecommon among the unemployed miners. The miners reported a statisticallysignificant improvement in grip force (p=0.031). They had asignificantly higher prevalence of symptoms associated withmining related exposures when compared with the population controls;pain in the upper extremities [relative risk (RR)=2.27, 95%confidence interval (Cl)=1.44–3.59), back pain (RR=1.84;Cl=1.237–2.75), vasospastic disease of the fingers (RR=2.05;Cl=1.18–3.57) and obstructive respiratory symptoms (attacksof dyspnea and wheezing: RR=3.67; Cl=1.167–11.6).     

Excès de cas humains et animaux de tularémie en France au cours de l’hiver 2007–2008 : émergence ou phénomène isolé ?

Context

Tularemia is a zoonosis affecting humans and hares in France. We describe the results of surveillance in both species, in 2007 and 2008.

Methods

Human tularemia cases are mandatorily notifiable in France since 2003. In hares, surveillance relies on volunteer hunter associations in all districts of the country. Data from mandatory reports and volunteer surveillance in 2007/2008 were analyzed and compared with previous results.

Results

In 2007/2008, 144 cases were reported in humans and 117 cases in hares. This was a 100% increase compared to previous years. Human cases differed from those of previous years only by the frequency of contact with breeding animals. Human cases without any documented risk exposure were also more frequent.

Conclusion

An increase of tularemia cases occurred in 2007/2008 in both species. Complementary studies are needed to identify the species reservoir in France to understand the causes of this peak of cases.     

Occupational health hazards in mining: an overview

This review article outlines the physical, chemical, biological, ergonomic and psychosocial occupational health hazards of mining and associated metallurgical processes. Mining remains an important industrial sector in many parts of the world and although substantial progress has been made in the control of occupational health hazards, there remains room for further risk reduction. This applies particularly to traumatic injury hazards, ergonomic hazards and noise. Vigilance is also required to ensure exposures to coal dust and crystalline silica remain effectively controlled.     

Manufacture and use of nanomaterials: current status in the UK and global trends

This paper provides an overview of the production and use of nanomaterials (NMs), particularly in the UK. Currently, relatively few companies in the UK are identifiable as NM manufacturers, the main emphasis being the bulk markets in metals and metal oxides, and some niche markets such as carbon nanotubes and quantum dots. NM manufacturing in the UK does not reflect the global emphasis on fullerenes, nanotubes and fibres. Some assumptions have been made about the types of NM that are likely to be imported into the UK, which currently include fullerenes, modified fullerenes and other carbon-based NMs including nanotubes. Many university departments, spin-offs and private companies have developed processes for the manufacture of NMs but may only be producing small quantities for research and development (R&D) purposes. However, some have the potential to scale up to produce large quantities. The nanotechnology industry in the UK has strong R&D backup from universities and related institutions. This review has covered R&D trends at such institutions, and appropriate information has been added to a searchable database. While several companies are including NMs in their products, only a few (e.g. manufacturers of paints, coatings, cosmetics, catalysts, polymer composites) are using nanoparticles (NPs) in any significant quantities. However, this situation is likely to change rapidly. There is a need to collect more information about exposure to NPs in both manufacturing and user scenarios. As the market grows, and as manufacturers switch from the micro- to the nanoscale, the potential for exposure will increase. More research is required to quantify any risks to workers and consumers.     

深圳公立医院管理体制改革制度设计

深圳公立医院管理体制改革实行政事分开、管办分开,在理事会架构下按法定机构模式组建市医管中心,落实公立医院运营管理自主权.作者从当前公立医院管理体制的弊端入手,介绍了深圳市进行公立医院管理体制改革的基本思路及改革方案设计的主要举措,深入剖析了的改革方案的特点,并对改革效果进行了预测.     

Effects of nitrogen dioxide on red blood cells of rats: Changes in components of red cell membranes during in vivo exposure to NO2

Red cell membranes, prepared from red blood cells of rats exposed to 4, 10, or 20 ppm nitrogen dioxide (NO₂) for 1 to 10 days, were examined for evidence of changes in membrane components. Appreciable changes were not found in contents of phospholipid and cholesterol during exposure to 10 ppm NO₂. By contrast, protein content altered with the time of exposure. Moreover, changes in protein composition were observed by employing sodium dodecyl sulfate — polyacrylamide gel electrophoresis. Twenty-four-hour exposure to NO₂ at the concentration above 10 ppm resulted in a marked increase in the percentage of lysophosphatidylethanolamine (LysoPE) to the total phospholipids. The prolonged exposure to 10 ppm NO₂ gave rise to a further increase in LysoPE, whereas the percentage of phosphatidylethanolamine (PE) showed a gradual decrease. A 1-day exposure to 4.0 ppm NO₂ also caused an increase in sialic acid content and decreases in those of PE and hexose. In addition to contents of these components the percentage of LysoPE increased 5 days after exposure and the elevated values were maintained up to the end of exposure period. These results demonstrate that red blood cells in circulation exhibit different membrane properties in terms of lipid and carbohydrate composition during 10 days of exposure to 4.0 ppm NO₂.     

Investigating clusters in the workplace and beyond

Clusters of disease are common and occur in the workplace and in the general community. They often arouse considerable concern among the population. Investigations have sometimes lead to exciting new knowledge, but in general the investigation of clusters is difficult and often unrewarding, especially for community clusters. In the workplace, investigations are more likely to find associations and even new causes, but still many clusters remain enigmatic. Despite this, there are many reasons for investigating clusters, including allaying community concern and identifying uncontrolled exposures. A structure for investigating clusters in the workplace is suggested.