两种菌体前处理方法对MALDI-TOF MS鉴定临床分离菌效果的影响

目的评价2种不同前处理方法对基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ ionization time of flight mass spectrometry,MALDI-TOF MS)鉴定临床分离菌效果的影响。 方法收集临床菌株43株,划线传代后,分别使用提取法和抹板法对微生物样本进行前处理,然后用MALDI-TOF MS鉴定比较两种处理方法的准确率和蛋白指纹图谱特征。 结果以16S rRNA/ITS RNA测序结果为参考标准,待测菌株经提取法处理后,MALDI-TOF MS鉴定革兰阳性菌、革兰阴性菌和真菌的种水平准确率分别为93.8%、100.0%和30.8%,待测菌株经抹板法处理后,MALDI-TOF MS鉴定革兰阳性菌、革兰阴性菌和真菌的种水平准确率分别为75.0%、100%和7.7%。提取法处理菌株后得到的蛋白指纹图谱中特征峰更多,信噪比更高,蛋白指纹图谱重现性更好。 结论推荐采用提取法为MALDI-TOF MS鉴定的常规前处理方法,其对建立标准的微生物检测流程,提高质谱鉴定的正确率,有重要价值。     

MALDI-TOF MS在儿童血培养阳性病原菌快速鉴定中的应用

目的 建立快速、准确的儿童血培养阳性病原菌鉴定方法,缩短血流感染鉴定时间,评估临床试验可行性.方法 运用分离胶法提取阳性血培养瓶中富集的菌落,利用基质辅助激光解析电离飞行时间质谱分析(MALDI-TOF MS)对经过一系列前处理后的血培养阳性标本进行鉴定,并与病原菌纯培养鉴定后的结果对比,比较MALDI-TOF MS快...     

相对定量质谱技术检测血清标志物方法部分关键点的优化对比和探讨

目的对iTRAQ结合质谱相对定量检测血清标志物实验关键点进行优化。方法采用正常人血清样本,对iTRAQ-LC-MS/MS检测血清标志物实验过程中,多种低丰度蛋白除盐浓缩方法、低丰度蛋白定量方法以及点靶仪反相洗脱效果进行比较。结果三种除盐浓缩方法中,蛋白除盐柱处理后所得蛋白得率最高(P0.01);五种蛋白定量方法中,Nanodrop2000测试仪测定低丰度蛋白的方法耗时最少,偏离度最小(P0.01);两种反向C18分离柱中,Magic柱洗脱效果更佳。结论优化后的实验条件有利于提高血清样本中蛋白的检出率。     

建立MALDI-TOF MS鉴定的海洋致病性弧菌数据库样本前处理的适宜条件

目的探讨建立基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ ionization time of flight mass spectrometry,MALDI-TOF MS)鉴定海洋致病性弧菌数据库前样本处理的适宜条件,为完善MALDI-TOF MS鉴定该菌入数据库提供参考。 方法将32株海洋致病性弧菌培养至24、48、72 h时,分别用直接转移法、扩展的直接转移法、甲酸提取法得到上机样品,然后用MALDI-TOF MS进行采图鉴定,用SPSS20.0软件进行统计学分析,对样本不同培养时间及3种处理方法的鉴定结果进行比较评价。 结果培养24 h鉴定分数平均值为2.321,属水平鉴定正确率为96.88%,种水平鉴定正确率为90.63%;培养48 h鉴定分数平均值为2.315,属水平鉴定正确率为93.75%,种水平鉴定正确率为90.63%;培养72 h鉴定分数平均值为2.316,属水平鉴定正确率为93.75%,种水平鉴定正确率为87.50%,3种不同培养时间的前处理方法鉴定至属(P=0.129)、鉴定到种(P=0.078),差异均无统计学意义。直接转移法鉴定分数平均值为1.986,属水平鉴定正确率为78.13%,种水平鉴定分数为59.38%;扩展的直接转移法鉴定分数平均值为2.135,属水平鉴定正确率为90.63%,种水平鉴定正确率为78.13%;甲酸提取法鉴定分数平均值为2.306,属水平鉴定正确率为96.88%,种水平鉴定分数为90.63%,3种不同上机前样本处理方法鉴定到属时差异有统计学意义(P=0.006);鉴定到种时差异有统计学意义(χ²=8.691,P=0.013)。 结论建立MALDI-TOF MS海洋致病性弧菌数据库时,选择培养时间24 h的甲酸法提取菌体蛋白的前处理方法较适宜,可得到高质量的蛋白指纹图谱和鉴定正确率。     

MassARRAY技术高通量检测乙肝病毒C区/C启动子区基因变异

目的建立基于MALDI-TOF质谱技术的高通量检测乙型肝炎病毒耐药基因变异的临床检测方法。方法应用MassARRAY Assay Design软件设计乙肝病毒前C区/C启动子区(BPC)iPLEX引物,进行PCR反应、虾碱性磷酸酶(SAP)反应,单碱基延伸反应,树脂脱盐与质谱分析。采集31例拉米夫定反应与17例拉米夫定耐药乙肝患者血清标本进行分析,并对MALDI-TOF质谱所检测的HBV基因变异区域进行DNA毛细管电泳测序比对。结果建立了基于MAL-DI-TOF质谱技术的HBV基因突变检测平台,实现了临床血清标本的高通量检测。31例拉夫米定反应患者均无HBV变异,17例拉米夫定耐药血清标本中乙肝病毒前C区/C启动子区(BPC)均检测到变异。MASSARRY检测结果与DNA测序结果一致。结论 MALDI-TOF质谱技术灵敏度高、准确度高,可高通量、快速检测HBV基因变异。     

出国体检人员中可疑氯胺酮阳性病例确证分析

目的建立一种快速、灵敏的方法用于氯胺酮初筛为阳性后的确证。方法采用传统的液-液萃取法进行人体尿液样品前处理,并结合单四极杆气相色谱质谱联用法(GC/MS)对样品进行确证分析。结果该氯胺酮初筛阳性尿样在确证分析中未检出氯胺酮。被检尿样的GC/MS谱图中未见明显的氯胺酮色谱峰;而经质谱定性后发现谱图中有较高的麻黄素色谱峰。结论本研究建立的GC/MS分析方法对于毒品确证十分准确、有效,可推广应用于其它毒品初筛为阳性的进一步确证实验中。     

超高效液相色谱-电喷雾串联四极杆质谱法测定高温烘烤食品中丙烯酰胺的方法研究

目的:建立超高效液相色谱-电喷雾串联四极杆质谱联用(UPLC/MS/MS)技术检测高温烘烤食品中痕量、超痕量的丙烯酰胺.方法:样品经提取净化,采用UPLC/MS/MS法,ESI( )电离方式,多反应监测(MRM)定量,考察和优化样品前处理方法、色谱条件、质谱参数等,验证回收率、精密度等,以建立理想的检测方法.结果:丙烯酰胺在1～100μg/L浓度范围内呈良好的线性,相关系数r=0.999606.高、中、低3种浓度平均回收率分别为:107.2%、103.7%、111.2%,相对标准偏差分别为:3.8%、2.7%、3.0%,均小于5%(n=5),检出限(LOD)为0.1μg/kg.结论:该方法快速,准确、灵敏,适用于高温烘烤食品中痕量、超痕量丙烯酰胺的检测.     

液相色谱-串联质谱法临床样品前处理专家共识

液相色谱-串联质谱法(LC-MS/MS)集色谱技术的高分离能力和质谱技术的高选择性、高特异性及高灵敏性的优点于一体, 成为临床检验领域最具有生命力的新技术之一, 但其分析效果往往受待测样品特性制约。由于质谱仪的抗干扰能力有限, 生物样本多需要进行合适的样品前处理才能有效提高检测性能, 实现精准检测。前处理的主要作用是将目标分析物从生物基质中有选择性地分离出来, 减少其他基质组分的干扰。同时, 还可将目标分析物浓缩和富集, 提高检测灵敏度。目前, 临床样本前处理方法不仅种类多, 个别方法耗时繁琐, 给实验室人员在方法选择和开发及标准化操作等方面带来了困难。因此, 本共识旨在为实验室方法建立提供指导, 助力临床质谱检测方法的研发规范发展。     

GC/MS法鉴定饮用水中的灭多威

目的：建立以气相色谱-质谱联用技术对多份水样进行灭多威成分鉴定的方法。方法：采用气相色谱-质谱联用法（GC／MS）对饮用水中灭多威进行定性分析，并比较样品处理中3种溶剂的提取效果。结果：灭多威在本次实验设置的色谱条件和质谱条件下能与其他有机物良好分离，在质谱图上可通过灭多威的105、58、88等几个特征离子峰进行准确定性；经比较发现二氯甲烷的提取效果较好。结论：GC／MS法鉴定未知有机毒物简单、灵敏、快速、准确，适合于公共卫生突发事件的应急处理。     

动物性食品中4种β-兴奋剂的超高效液相色谱-串联质谱法测定

目的利用超高效液相色谱-串联质谱(UPLC-MS/MS)测定动物性食品中克伦特罗、莱克多巴胺、沙丁胺醇和特布他林。方法样品经葡萄糖醛甙酶/芳基硫酸酯酶酶解,乙酸乙酯提取,固相萃取小柱净化后UPLC-MS/MS测定。实验优化了液相条件、质谱参数和固相萃取前处理步骤。结果 4种瘦肉精在浓度为1.0μg/L~25.0μg/L时,线性关系较好,相关系数(r)为0.999。莱克多巴胺、克伦特罗、沙丁胺醇、特布他林的检出限均为0.5μg/kg。当加标水平为2.0μg/kg时,回收率为84%~105%。结论本法可用于实际样品的检测,样品中莱克多巴胺的检出率为5.0%。     

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.     

The human drama of genetics: 'hard' and 'soft' media representations of inherited breast cancer

This paper examines media coverage of 'breast cancer genetics', and explores its implications for public understanding. We present a content analysis of coverage in British newspapers and look at a variety of popular forms, including women's magazines, television soap opera and radio drama. Genetic/inherited risk receives a great deal of coverage across a wide range of media formats and outlets. Much of this attention has focused on individuals from 'high risk families' and dilemmas around prophylactic mastectomies. Through examining media coverage, combined with interviews with media personnel and their sources, we show why this story proved so attractive to the media and highlight the different production values which influence coverage. Finally, we introduce preliminary findings from focus group discussions to demonstrate how such 'human interest' framing has engaged audience attention and influenced public understandings. The paper concludes by highlighting the implications for analysing, predicting, and engaging with, media representations of science.     

Worker education in the primary prevention of occupational dermatoses

This paper reports the evaluation of a skin care education programmeconducted on a fine chemicals manufacturing site where over1,000 employees are located. Approximately 60% are involvedin chemical manufacture. Over a 12 month period production staffreceived training in prevention of occupational dermatoses linkedto a site-wide poster initiative. The incidence of new casesof occupational dermatoses fell from 0.055 (70 cases in 1,277employees) to 0.021 (27 cases in 1,277 employees) before andafter the intervention respectively (p<0.0001). After otherfactors such as chemicals handled, observer bias and changesin reporting related to socioeconomic climate were taken intoaccount it is concluded that this study demonstrates the importanceof worker education as a tool for primary prevention of disease.Training materials such as video and poster presentations maybe effectively used in the chemical manufacturing industry asan adjunct to prevention and control of exposure to substanceshazardous to the skin. Such methods may also be used in otherindustries where there are significant risks of dermatoses.     

Regional Variation in Travel-related Illness acquired in Africa,March 1997–May 2011

To understand geographic variation in travel-related illness acquired in distinct African regions, we used the GeoSentinel Surveillance Network database to analyze records for 16,893 ill travelers returning from Africa over a 14-year period. Travelers to northern Africa most commonly reported gastrointestinal illnesses and dog bites. Febrile illnesses were more common in travelers returning from sub-Saharan countries. Eleven travelers died, 9 of malaria; these deaths occurred mainly among male business travelers to sub-Saharan Africa. The profile of illness varied substantially by region: malaria predominated in travelers returning from Central and Western Africa; schistosomiasis, strongyloidiasis, and dengue from Eastern and Western Africa; and loaisis from Central Africa. There were few reports of vaccine-preventable infections, HIV infection, and tuberculosis. Geographic profiling of illness acquired during travel to Africa guides targeted pretravel advice, expedites diagnosis in ill returning travelers, and may influence destination choices in tourism.     

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.     

The design of hazard risk assessment matrices for ranking occupational health risks and their application in mining and minerals processing

Two hazard risk assessment matrices for the ranking of occupational health risks are described. The qualitative matrix uses qualitative measures of probability and consequence to determine risk assessment codes for hazard-disease combinations. A walk-through survey of an underground metalliferous mine and concentrator is used to demonstrate how the qualitative matrix can be applied to determine priorities for the control of occupational health hazards. The semi-quantitative matrix uses attributable risk as a quantitative measure of probability and uses qualitative measures of consequence. A practical application of this matrix is the determination of occupational health priorities using existing epidemiological studies. Calculated attributable risks from epidemiological studies of hazard-disease combinations in mining and minerals processing are used as examples. These historic response data do not reflect the risks associated with current exposures. A method using current exposure data, known exposure-response relationships and the semi-quantitative matrix is proposed for more accurate and current risk rankings.     

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.     

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.