检测SARS冠状病毒的套式RT-PCR方法的建立与初步应用

[目的]建立套式RT-PCR检测SARS冠状病毒的方法。[方法]从广东地区SARS患者尸解肺组织、咽拭子、嗽口水及本实验室分离的SARS冠状病毒（SAPS—CoV）组织培养上清中提取RNA，利用针对SARS冠状病毒多聚酶基因的特异性引物，进行逆转录及套式PCR扩增。扩增出的阳性片段连接入pGEM—T载体中，测序后比较其与其他已知SARS冠状病毒的同源性。[结果]从SARS患者尸解肺组织、咽拭子、嗽口水及SARS冠状病毒组织培养上清中均可扩增出阳性片段，经测序后证实是SARS冠状病毒特有序列。[结论]针对SARS冠状病毒多泵酶基因所建立的套式RT—PCR方法适用于临床标本及组织培养标本中SARS冠状病毒的检测。     

传染性非典型肺炎疑似病例实验室快速诊断的初探

目的 对传染性非典型肺炎(SARS)疑似病例进行实验室快速诊断。方法 采集SARS疑似病例血清，先对标本进行病毒颗粒浓缩，再用特异性引物进行Nested RT—PCR，检测SARS冠状病毒RNA；另外，采用免疫荧光法进一步同时检测血清中9种呼吸道病原体(嗜肺军团茵血清型1、肺炎支原体、Q热立克次体、肺炎衣原体、腺病毒、呼吸道舍胞病毒、甲型流感病毒、乙型流感病毒和副流感病毒)IgM抗体。结果 对21倒SARS疑似病例血清进行Nested RT—PCR，结果均未扩增出目标条带。进一步对21例患者血清进行同步检测血清中9种病原体IgM抗体，共有14例患者甲型流感病毒IgM抗体阳性，阳性率为67％。结论 对SARS疑似病例进行SARS冠状病毒RNA和其他9种呼吸道病原体IgM抗体检测是实用可行的。     

广东省传染性非典型肺炎病例的SARS冠状病毒分离株的分子生物学鉴定

目的 对广东省部分传染性非典型肺炎(SARS)病例的SARS冠状病毒分离株进行鉴定，并初步建立SARS冠状病毒PCR检测方法。方法 采集临床诊断为SARS病例的咽漱液标本，进行多种细胞分离培养，对出现病变的细胞分离物采用冠状病毒特异引物通过逆转录聚合酶链反应(RT-PCR)及巢式聚合酶链反应(Nested—PCR)进行扩增，并对部分扩增片段进行序列测定，应用DNASTAR软件分析比较oN时采用间接免疫荧光(IFA)和ELISA法检测上述患血清中SARS冠状病毒Igc抗体。结果 对21例SARS患的细胞分离物进行PCR扩增，结果均阳性，其中12份患咽漱液标本PCR扩增结果也阳性，序列测定及基因分析表明，广东省SARS病例的病毒分离株为冠状病毒，其基因序列与WHO公布的SARS基因序列一致，氨基酸序列与目前已知的冠状病毒同源性为58％～76％。时对21例患进行血清学检测，其中IFA检测有9例阳性，ELISA检测有12例阳性。结论 从广东省部分SARS病例标本中分离的病毒株是一种新的冠状病毒，PCR检测具有早期、快速的优点。     

人副流感病毒HPIV多重RT—PCR快速检测方法的建立

目的建立快速检测人副流感病毒的多重PCR方法。方法通过查阅文献获取副流感病毒的PCR引物序列，并利用分子生物学软件对其进行评估，同时设计合成半巢式PCR引物。提取分离病毒的总RNA，以RNA抽提物为模板，一步法一次反转录扩增HPIV-1，2，3，4等4种型别的副流感病毒。以多重RT—PCR产物作为模板，用半巢式PCR进一步扩增确认。结果利用多重RT—PCR方法一次扩增出人副流感病毒的4个不同型别，条带大小分别是317、507、189和451bp，与目的片段大小一致；半巢式PCR扩增出相应的特异条带，条带大小分别是261、340、145和390bp，扩增结果与目的片段大小一致。结论人副流感病毒多重RT—PCR快速检测方法已初步建立。     

RT-PCR检测病媒生物SARS冠状病毒结果初报

目的调查广东重点地区鼠类和蜚蠊是否携带SARS冠状病毒或者是否SARS冠状病毒的宿主，为寻找SARS冠状病毒的来源及控制SARS流行提供依据。方法巢式RT—PCR技术、荧光定量RT-PCR技术。结果利用SARS冠状病毒特异性引物对广州、中山、江门及恩平等地的160份鼠肺样品及15份蜚壕的体表擦拭子检测结果表明，荧光定量RT—PCR未检测到阳性，巢式RT—PCR显示来自广州市的1份蜚蠊体表擦拭子呈可疑阳性。结论巢式RT、PCR、荧光定量RT-PCR都适合于病媒生物SARS冠状病毒的初步筛选，目前未有明显证据显示鼠类及蜚镰携带和传播SARS冠状病毒,仍需作进一步研究。     

登革2型病毒全长基因组的长链RT-PCR法扩增

目的采用长链RT—PCR技术扩增登革2型病毒新几内亚株（NGC）全长基因组。方法根据登革2型病毒NGC株基因组全序列设计引物，在上游引物中加入Sp6RNA聚合酶启动子核心序列，下游引物中加入Cla Ⅰ内切酶位点。从病毒感染的乳鼠脑中提取RNA，采用长链RT—PCR技术进行扩增。以获得的PCR产物为模板分别扩增3个NGC株特异的基因片段。结果长链RT—PCR法扩增出约11kb基因片段，经PCR法证实为登革2型病毒NGC株特异序列。结论通过长链RT—PCR技术，获得了登革2型病毒NGC株全长基因组，为进一步构建感染性克隆打下基础。     

逆转录-聚合酶链反应在HIV-1感染诊断中的应用

[目的]评价逆转录-聚合酶链反应(RT—PCR)在HIV—l感染诊断中的应用；[方法]采用RNA一步法试剂盒提取RNA。用3套分别来自于LTRgag、pol、env不同区域的序列引物进行嵌套式PCR扩增。[结果]对24例血清标本进行检测，其中15例为HIV感染者ll例为免疫印迹试验可疑待确定，但经过1年的随访可排除；另外8例为正常者。经过巢式扩增，15份阳性标本中有14份扩增出两条或两条以上核酸片段，l份未扩增出任何一条片段，其敏感性达93．3％(14／15)。其中6份肝素抗凝标本env区域均未扩增出片段。阴性标本无片段，其特异性为100％(8／8)。[结论]RT-PCR具有高敏感性和特异性，可用于窗口期HIV感染的诊断及HIV—l阳性母亲所生婴儿的HIV检测，可作为免疫印迹试验的补充和辅助诊断。     

2例马来西亚籍输入性基孔肯雅热病例的实验室诊断

[目的]通过系统的实验室检测发现并确认2例输入性基孔肯雅病例。[方法]采用实时荧光逆转录（Realtime RT-PCR）、逆转录PCR（RT—PCR）检测方法对病人血清进行检测,并对RT-PGR扩增产物进行核苷酸序列测定。[结果]通过实时荧光RT—PCR、RT—PCR 2种实验方法在该病例标本中检出基孔肯雅病毒核酸;对RT-PCR扩增产物进行测序,测出的521个碱基与基孔肯雅病毒核酸序列比对,结果同源性高达99％。[结论]2病例为输入性基孔肯雅实验室确诊病例。     

实时聚合酶链反应检测O1群和O139群霍乱弧菌方法的建立及应用

目的建立检测O1群和O139群霍乱弧菌的实时荧光聚合酶链反应（RT—PCR）方法并进行水体标本的检测评价。方法根据O1群和O139群霍乱弧菌O抗原编码基因rfb序列设计引物，利用SYBRGreen染料，建立同时检测霍乱弧菌O1群和O139群的RT—PCR方法，对所建立的方法分别进行实验室内的灵敏度、特异性及重复性评价。采集河口水标本增菌后进行RT—PCR检测，与分离培养方法比较评价实际应用价值。结果建立了检测O1群和O139群霍乱弧菌的双重RT—PCR方法，根据扩增产物的溶解温度能有效区分O1群和O139群霍乱弧菌两种目标片段的扩增；对其他10种弧菌染色体DNA没有扩增；RT—PCR检测524份河口水体标本的增菌液，与常规分离方法相比显示了明显的灵敏性，并且所有常规分离方法阳性标本其荧光PCR检测亦为阳性。结论以O1群和O139群rfb基因为目标检测片段建立的霍乱弧菌RT—PCR方法可用于环境水体样本中霍乱弧菌常规分离前的快速筛查。     

广东省SARS冠状病毒分离株S基因全序列分析

目的 测定并分析广东省传染性非典型肺炎病人标本中分离到的严重急性呼吸综合征(SARS)冠状病毒的S基因全序列。方法 选取3株来自中山市、广州市、江门市传染性非典型肺炎病人的SARS冠状病毒分离株，设计8对相互嵌套的S基因特异引物扩增病毒的S基因，直接进行PCR产物测序，将所测的序列与从GemBank上获得的世界各地10株SARS冠状病毒分离株的S基因序列用DNASTAR软件包进行基因变异分析。结果 3株毒株经P(R扩增后，均可获得大小分别为513、589、527、509、621、501、578、678bp的基因片段，测序并拼接可获得一长3768个碱基S基因全基因序列，编码1256个氨基酸；基因序列比对结果显示，与世界各地10株毒株比较，核苷酸和氨基酸的最大同源性在99．7％～100．0％和99．2％～99.9％之间，其变异主要发生在S蛋白的S1片段。结论 SARS冠状病毒的S蛋白是比较保守的．但S1片段上某些位点的变异可能是病毒的毒力不同的主要原因。     

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.