单管多重荧光定量RT-PCR方法鉴别A组轮状病毒及诺如病毒GⅠ型和GⅡ型

目的建立能同时检测A组轮状病毒、诺如病毒GⅠ型和GⅡ型的多重荧光定量RT-PCR方法。方法分别选用轮状病毒NSP3基因、诺如病毒GⅠ型和GⅡ型orf1与orf2基因中的64 bp、90 bp和185 bp片段作为荧光定量PCR检测的靶标,设计引物和Taq Man探针,构建标准品,绘制标准曲线,评价检测方法的重复性、特异度和灵敏度,并对138份粪便标本进行检测及测序验证。结果 3种病毒对应的标准曲线分别为:轮状病毒:y=-3.9lgx+41.204;诺如病毒GⅠ型:y=-3.06lgx+40.716;诺如病毒GⅡ型:y=-3.29lgx+41.538,质粒标准曲线的方差系数为0.997,灵敏度达到每个反应102copy/μl,特异度强。138份样本中检出A组轮状病毒、诺如病毒GⅠ型和GⅡ型的检出率分别为32.6%、5.8%和26.1%,与基因测序比对结果相符。结论构建可用于A组轮状病毒及诺如病毒GⅠ型和GⅡ型的多重荧光定量RT-PCR方法快速、特异且灵敏,可用于临床病原诊断和流行病学调查。     

双重实时荧光定量PCR法检测腹泻患者粪便中诺如病毒(GⅠ+GⅡ)的研究

目的建立同时检测GⅠ和GⅡ型诺如病毒的双重实时荧光定量PCR方法,并在临床检验中得到应用。方法本研究采用了双重实时荧光定量PCR法,对诺如病毒GⅠ型病毒的特异性Taqman探针的5’端标记了FAM和对诺如病毒GⅡ型病毒的特异性Taqman探针的5’端标记了JOE,从而实现了在同一反应管中对诺如病毒同时进行检测和分型。2015年采集两所哨点医院符合要求的门诊腹泻患者的380份粪便标本运用此方法进行了检测和分型。结果诺如病毒GⅠ型和诺如病毒GⅡ型分别在FAM检测通道和JOE检测通道出现相应的荧光扩增曲线;380例符合定义的临床标本中诺如病毒GⅠ型的检出率为3.68%,诺如病毒GⅡ型的检出率为14.74%,诺如病毒GⅠ型和诺如病毒GⅡ型混合感染的检出率为1.58%,全年诺如病毒的总检出率为20.00%。结论双重实时荧光定量PCR法具有重复性好、特异性强、时效性好和简单易操作的特点,具有极大的应用和推广价值。     

荧光定量RT-PCR检测诺如病毒基因Ⅱ型方法的建立和评估

目的 建立灵敏、特异的诺如病毒基因Ⅱ型的荧光定量RT-PCR方法,用于临床腹泻粪便样本病毒的定量检测.方法 构建质粒DNA,并转录合成RNA作为标准品,建立和优化诺如病毒基因Ⅱ型荧光定量RT-PCR方法和反应体系,评价该方法的灵敏度、特异性、重复性,并进行临床粪便样本的检测.结果 此方法最低可以检出102拷贝数/μl,能特异地检出诺如病毒基因Ⅱ型,与诺如病毒基因Ⅰ型无交叉反应,与柯萨奇病毒B组、脊髓灰质炎病毒、肠道病毒、星状病毒、甲肝病毒、埃可病毒和轮状病毒无交叉反应.针对标准品,2次批内试验的荧光信号循阈值(Ct值)变异系数(CV)分别为1.60％、0.70％,2次批间试验的变异系数(CV)分别为0.40％、0.40％,均在5％以下.结论 本研究建立的实时定量RT-PCR检测诺如病毒基因Ⅱ型的方法,其灵敏度、特异性和重复性良好,可用于该病毒的快速检测.     

GⅠ和GⅡ型诺如病毒双重荧光RT-PCR法检测

目的 建立应用Allglo探针同时检测GⅠ和GⅡ型诺如病毒的双重荧光反转录\聚合酶链反应(RT-PCR)方法.方法 设计针对GⅠ和GⅡ型诺如病毒开放阅读框架ORF1及ORF2为目的基因的引物和Allglo探针,优化最佳反应条件,建立一步法荧光RT-PCR快速检测反应体系;对该方法的特异性、抗干扰性、灵敏度进行评估,对96份临床粪便标本进行检测、测序分析.结果 该方法特异性强,与轮状病毒、扎如病毒、星状病毒、腺病毒同时检测无交叉反应;同一体系下GⅠ或GⅢ型诺如病毒相互之间没有干扰;最低检测限均为10 copies/μL;对96份临床粪便标本进行检测,结果与单重荧光RT-PCR方法符合率100％,且测序结果显示目标序列正确.结论 本研究建立的Allglo探针法检测GⅠ和G Ⅱ型诺如病毒技术特异性好,灵敏度高,可用于感染性腹泻暴发中诺如病毒的快速筛查.     

实时逆转录环介导等温扩增检测GⅡ.4型诺如病毒的研究

目的建立快速检测诺如病毒GⅡ.4型的实时逆转录环介导等温扩增方法(RT-LAMP)。方法选取GⅡ.4型诺如病毒的ORF1与ORF2接头处较为保守的基因序列设计RT-LAMP引物并进行筛选,优化反应条件,分析方法的灵敏度、分析特异性和稳定性,采用优化的方法检测40例临床病毒性腹泻样本,结果与实时荧光逆转录PCR(RT-PCR)比较。结果该研究建立的诺如病毒GⅡ.4型RT-LAMP的最佳反应温度为65℃;该方法检测GⅠ型和GⅡ非诺如病毒4型、轮状病毒、札如病毒、星状病毒、腺病毒、CA16、EV71、CA6和CA10等常见10种其他腹泻病毒的结果为阴性;该方法对诺如病毒GⅡ.4型的检测能力与实时荧光RT-PCR相当;与实时荧光RT-PCR相比,该研究建立的RT-LAMP法检测诺如病毒GⅡ.4型阳性样本和诺如病毒阴性样本的符合率为100%。结论该研究建立的诺如病毒GⅡ.4型RT-LAMP快速检测方法灵敏度高,特异性好,为建立诺如病毒其他基因型的RT-LAMP检测方法奠定了基础。     

实时荧光定量PCR联合检测诺如病毒和副溶血性弧菌的方法建立与应用

目的建立实时荧光定量PCR法联合检测诺如病毒和副溶血性弧菌的方法。方法根据GⅠ型、GⅡ型诺如病毒和副溶血性弧菌tlh基因设计特异性引物。通过调整引物浓度和优化反应条件,建立多重荧光定量PCR检测体系,对方法的灵敏度和特异性进行验证。结果多重荧光定量PCR检测方法对诺如病毒、副溶血性弧菌、轮状病毒、沙门菌、志贺菌、大肠杆菌O157等样本进行检测,未发生交叉反应。结论本研究建立的实时荧光定量PCR法同时检测诺如病毒和副溶血性弧菌灵敏度高、特异性好,能用于食品携带病原微生物和感染性腹泻暴发中诺如病毒和副溶血性弧菌的检测。     

RT-LAMP法对Ⅱ型诺如病毒的检测

目的在本实验室建立适合于基层使用的Ⅱ型诺如病毒快速检测方法 RT-LAMP法,并将其应用于临床病毒性腹泻的检测,为了解诺如病毒导致的病毒性腹泻在南京市的流行状况提供方法和数据。方法用实验室保存的诺如病毒阳性标本进行方法学建立,并收集南京地区某医院2011年秋冬因腹泻就诊并临床诊断为病毒性腹泻的57例成年人患者的粪便进行Ⅱ型诺如病毒检测,包括荧光定量PCR法及基于环的等温扩增(LAMP)法,并对检测为阳性标本核酸进行测序分析。结果所建立的Ⅱ型诺如病毒RT-LAMP法和荧光定量检测法均可以检测到稀释到10 000倍的阳性标本,即两种方法具有相同的敏感度。对于诺如病毒I型、甲肝病毒及乙肝病毒没有扩增,即对诺如病毒Ⅱ型的扩增具有特异性。57例临床粪便标本,荧光定量PCR法与RT-LAMP法均检测到6例Ⅱ型诺如病毒阳性病例,两种方法符合率为100%,经测序分析,5例为诺如病毒GⅡ.4亚型,1例为诺如病毒GⅡ.6亚型。5例GⅡ.4亚型分别来源于4个不同的流行株,3个国家和地区,巴西、俄罗斯和北京。结论南京地区2011年诺如病毒腹泻病例以GⅡ.4亚型为主要感染亚型,也有GⅡ.6亚型。结果提示该病毒传播范围的广泛性和流行病学防控的艰巨性。荧光定量PCR与RT-LAMP法都可以快速进行病原检测,但RT-LAMP法因其方法简易、不需要特殊仪器、结果直接肉眼观察而更适合基层使用。     

诺如病毒快速检测方法的建立及其在国境卫生检疫中的应用

目的:建立快速、特异的诺如病毒实时荧光RT-PCR与常规RT-PCR相结合检测方法,对人感染诺如病毒进行快速检测。方法:根据国外最新流行的诺如病毒核酸序列,设计并合成诺如病毒基因扩增引物及荧光标记探针,摸索最佳反应条件,制定出快速、灵敏检测诺如病毒核酸的实时荧光RT-PCR检测方法及常规RT-PCR检测方法。结果:实验证明,本方法将实时荧光RT-PCR与常规RT-PCR检测方法相结合,能在5 h内快速确认诺如病毒,检测灵敏度达到102拷贝/反应,具有快速、灵敏的特点,且具有较高的特异性和重复性。结论:本实验室利用本方法,首次在广州某国境口岸从外籍发热船员样本中检出5例输入性GGⅡ群诺如病毒。该方法对防止诺如病毒传入我国,保障我国国境卫生安全具有重要意义。     

感染性腹泻疫情检测结果分析

目的明确一起感染性腹泻疫情的病原体和来源。方法通过流行病学调查,查明罹患率。采集疑似感染性腹泻患者肛拭子、生活饮用水、空气和环境涂抹拭子,用实时荧光定量RT-PCR法快速检测轮状病毒A群、B群、C群,诺如病毒GⅠ、GⅡ型和肠道腺病毒三种感染性病毒核酸,肛拭子采用细菌培养检测肠道致病菌,生活饮用水检测菌落总数和大肠菌群。结果共采集病例标本21份,生活饮用水2份,厕所空气样2份,环境标本标本厕所蹲位地面墙面、寝室门把手地面等涂抹拭子20份,病例标本13份、环境标本3份为诺如病毒核酸GⅡ型,其他轮状病毒A群、B群、C群,诺如病毒GⅠ型和肠道腺病毒核酸、肠道致病菌检测为均阴性,饮用水菌落总数和大肠菌群检测均未检出。结论该起疫情为诺如病毒感染所致。暴发的可能传播为人与人接触传播和空气气溶胶传播。实时荧光定量RT-PCR法准确快速的检测出诺如病毒GⅡ型病原体,为疫情的处理提供了及时正确的方向。     

应用实时荧光定量RT-PCR技术快速检测基因Ⅱ型诺如病毒

目的建立基因Ⅱ型诺如病毒的实时荧光定量RT-PCR检测方法,并应用于临床标本的检测。方法二氧化硅法提取粪便中的病毒RNA,使用针对基因Ⅱ型诺如病毒N/S结构域的引物和探针建立荧光定量PCR方法,对罗城县急性胃肠炎疫情的34份标本进行检测,并与常规RT-PCR和ELISA检测结果比较;还用该法对2006年2月至2007年1月广西急性胃肠炎暴发及散发病例粪便样本305份做了临床检测。结果用实时荧光定量RT-PCR法成功地从罗城疫情样本中检测出基因Ⅱ型诺如病毒,实时荧光定量RT-PCR、常规RT-PCR和ELISA的检出率分别为为85.3%、76.5%和26.5%。而实时荧光定量RT-PCR对114份对急性胃肠炎暴发疫情及191份散发病例样本的基因Ⅱ型诺如病毒检出率分别为56.1%和23.6%。结论诺如病毒的实时荧光定量RT-PCR检测方法具有快速、灵敏和特异的优点,不但能对标本中病毒浓度进行定量检测,还能鉴别诺如病毒基因型别。同时也发现基因Ⅱ型诺如病毒感染在广西急性胃肠炎暴发疫情和散发病例中较常见。     

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.     

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.     

Psychopathological features of a patient population of targets of workplace bullying

BACKGROUND: A strong association between workplace bullying and subsequent anxiety and depression, indicated by empirical research, suggests that bullying is an aetiological factor for mental health problems. AIMS: To evaluate levels of stress and anxiety-depression disorder developed by targets of workplace bullying together with outcome at 12 months and to characterize this population in terms of psychopathology and sociodemographic features. METHODS: Forty-eight patients (36 women and 12 men) meeting Leymann Inventory of Psychological Terror criteria for bullying were included in a prospective study. Evaluations were performed at first consultation and at 12 months using a standard clinical interview, a visual analogue scale of stress, the Hospital Anxiety and Depression (HAD) scale, the Beech scale of stress in the workplace and a projective test (Picture-Frustration Study). RESULTS: At first consultation, 81% of patients showed high levels of perceived stress at work and 83 and 52% presented with anxiety or depression, respectively. At 12 months, only 19% of working patients expressed a feeling of stress at work. There was a significant change in symptoms of anxiety while there was no change in symptoms of depression. Stress at work and depression influenced significatively capacity to go back to work. At 12-month assessments, workers showed a significantly better score on the HAD scale than non-workers. Over half the targets presented a neuroticism-related predominant personality trait. CONCLUSION: Workplace bullying can have severe mental health repercussions, triggering serious and persistent underlying disorders.     

The rise and fall in incidence of malignant mesothelioma from a British Naval Dockyard, 1979-1999

BACKGROUND: The incidence of malignant mesothelioma in Britain is predicted to rise over the next 15-25 years because of past failure to protect the workforce against inhalation of asbestos. In British Naval dockyards, alternative insulation materials and respiratory protection were introduced from the mid-1960s. Aims This study was carried out to investigate the effects of these control measures on mesothelioma deaths in dockyard workers. METHODS: Cases of mesothelioma of the pleura and peritoneum between 1979 and 1999 in workers from the Devonport Naval Dockyard, south-west England, were sought from coroners' and medico-legal records. RESULTS: Three hundred and one cases were identified, 7% peritoneal. The peak incidence occurred in 1991 with 25 cases per annum (quadratic model fit R(2) = 74.2%, P < 0.001) and we predict that by 2003 the incidence will fall to fewer than five cases per annum. The mean time between first exposure and presentation was 48.5 years [95% confidence interval (CI) = 47.3-49.8], but this was significantly shorter in the more heavily exposed trades, when compared with the less heavily exposed (42 years, 95% CI = 39.0-45.0, versus 49.5 years, 95% CI = 48.2-50.9). Those with higher exposure were also at significantly greater risk of peritoneal disease (P < 0.023, Fisher's exact test). CONCLUSION: The reduction in incidence of mesothelioma is greater than can be accounted for by reduction in numbers of dockyard workers over the last 50 years. Changes in insulation materials and improved industrial hygiene measures introduced into the Devonport Dockyard from the mid-1960s have resulted in an earlier decline in the incidence of malignant mesothelioma than that predicted for the British workforce as a whole.     

Die sogenannte Vinylchlorid-Krankheit—eine berufsbedingte Systemsklerose?

Zusammenfassung Es wurden 13 Arbeiter untersucht, die zwischen 1 3/4 und 18 Jahren in einem PVC-herstellenden Betrieb beschäftigt waren. 8 von ihnen wiesen sklerodermieartige Hautveränderungen (Abb. 2) auf, die histologisch charakterisiert sind durch Verbreiterung und Homogenisierung der kollagenen Faserbündel (Abb. 3 u. 4) sowie Fragmentation und Rarefizierung der elastischen Fasern (Abb. 5). Bei 7 Patienten bestanden trommelschlegelartige Auftreibungen einzelner Fingerendphalangen (Abb. 1). 11 Patienten zeigten Durchblutungsstörungen der Extremitäten (4 von diesen ein Raynaud-Syndrom) und 6 Patienten Acroosteolysen einzelner Fingerendphalangen (Abb. 6, Tabellen 1–3).Darüber hinaus bestand bei allen Patienten eine Thrombocytopenie, bei 12 Patienten eine Splenomegalie, bei 11 Patienten eine Einschränkung der Leberfunktion (erhöhte BSP-Retention). An der Leber ließ sich histologisch bei den 5 laparoskopierten Patienten eine deutliche Fibrosierung der Portalfelder nachweisen. Bei 4 Patienten bestanden Oesophagusvaricen (Tabellen 4 u. 5). 8 Patienten zeigten eine Partialinsuffizienz mit Hinweisen für vorwiegend restriktive Veränderungen.Die hier beschriebenen Veränderungen gehen weit über das bisher bekannte Acroosteolysesyndrom hinaus. Es wird der dringende Verdacht geäußert, daß langfristige Vinylchlorid-Exposition dieses komplexe Krankheitsbild verursacht. Daher wird für diese systemische Krankheit die Bezeichnung Vinylchlorid-Krankheit vorgeschlagen.Wir danken Frau E. Voigtländer für die Herstellung der klinischen und histologischen Abbildungen.     

Organophosphate toxicity and occupational exposure

The ubiquitous organophosphates present a continuing health hazard in agriculture, public health eradication programmes and as chemical warfare agents. Despite significant progress in understanding the potential mechanisms of toxicity far beyond the commonly accepted mechanism of cholinesterase inhibition in intentional exposures, the precise health effects following occupational exposures are yet to be completely defined. A much greater understanding exists of the clinical features of organophosphate poisoning. These are characterized by a triphasic response involving an initial acute cholinergic phase, an intermediate syndrome (both associated with high mortality) and a disabling but non-lethal delayed polyneuropathy. The delayed polyneuropathy may occur in the absence of the cholinergic or intermediate phases. However, progress is still required in order to improve the quantification and assessment of occupational exposures and the implementation of appropriate preventive measures. Finally, evidence-based guidelines for appropriate or optimal therapeutic interventions following poisoning are required urgently and collaborative work with colleagues in developing countries, where the occurrence of organophosphate exposures is more frequent, may provide the answers.     

Miliaria rubra of the lower limbs in underground miners

This report documents a case series of miliaria rubra of the lower limbs in miners at a deep underground metalliferous mine in tropical arid Australia. During the summer months of February and March 1999, all cases of miliaria rubra of the lower limbs in underground miners seen at the mine's medical centre were clinically examined and administered a questionnaire. Twenty-five patients were seen, an incidence of 56.4 cases per million man-hours. Miliaria rubra was most often located between the ankle and knee (88% of cases). Twenty-four percent had concurrent folliculitis and 20% had concurrent tinea. Thirty-two percent had a personal history of asthma. Walking through ground-water and splashing of the legs was common. Three to 4 weeks of sedentary duties in air conditioning was generally required to achieve resolution of miliaria rubra. The incidence of miliaria rubra of the lower limbs is 38% of the incidence of heat exhaustion at the same mine. The length of disablement is greater, however. Atopics may be at increased risk of miliaria rubra. Control measures are discussed.     

Planning research

The starting point for any research project should be a question.Once this has been defined and the relevant scientific literaturereviewed, a protocol should be drawn up. This will be used notonly as a guide to the conduct of the study and in the preparationof the final report, but also in seeking any financial supportand approvals that are required for the investigation. A protocolis normally arranged in sections covering the background tothe study, the question(s) that it will address, the methodsthat will be used for the collection and analysis of data, thestatistical power of the investigation (where relevant), anyethical considerations, and the financial input that will beneeded. A pilot study is often helpful where aspects of thestudy method are untried or of uncertainvalidity.