多重PCR和单重PCR在流感病毒快速检测中的比较与应用

[目的]建立快速检测H1、H3亚型和乙型流感病毒的多重PCR技术,并与单熏PCR技术进行比较与应用. [方法]根据H1、H3亚型和乙型流感病毒设计引物,进行多重PCR扩增及电泳检测,在快速检测中与单重PCR进行比较与应用. [结果]该多重PCR技术能在4 h内同时检测3种型别的流感病毒,时间短于单重PCR技术,特异性和灵敏度相似.[结论]本研究建立多重PCR技术可以准确检测流感病毒,不仅灵敏度高、特异性好,而且可以得到快速的检测结果.     

香烟焦油对小鼠急慢性毒性实验研究

目的:观察香烟焦油对小鼠的急、慢性毒性作用。方法:提取香烟中的焦油,小鼠40只,分为染毒组,乙醇组和生理盐水组作对照,腹腔注射法(1次/d)染毒,连续4d,观察小鼠活动情况并记录。4d后,处死小鼠,取胸骨骨髓常规制片,观察骨髓PCE细胞微核发生率。另取60只小白鼠,方法相同,连续观察小鼠30d活动情况,最后1d解剖,观察小鼠内脏各器官颜色、体积等改变。结果:急性毒性实验小鼠出现活动能力下降、尿失禁等现象,而慢性毒性实验小鼠出现体重下降、皮肤松弛、活动减少、尾部溃疡等症状,解剖后可观察到染毒内脏各器官肺、肝、肾、胃肠道颜色、体积等改变。小鼠骨髓细胞微核表明,香烟焦油可诱导微核形成,与对照组比较差异有统计学意义(P<0.05)。结论:香烟焦油对小鼠有较强的毒性作用,可能会对人体产生相应的危害。     

甲型流感病毒血凝素基因变异与抗原变异关系研究

目的 了解甲型流感病毒流行株的血凝素基因变异和抗原变异及两者间的关系,结合流行病学资料分析基因变异和抗原变异的吻合情况.方法 从2005-2007年上海市季节性流感样病例分离到的甲型流感病毒株中,选取H1N1、H3N2两个亚型中部分有代表性的病毒株,并同WHO北半球流感疫苗推荐株一起,进行血凝素全基因序列测定后做基因进化树分析.同时用灭活的全病毒抗原免疫金黄地鼠,通过血凝抑制试验测定病毒的血凝效价,对血凝抑制结果进行聚类分析,绘制病毒的抗原变异图.结果 H3N2分离株与WHO北半球疫苗推荐株A/Sydney/5/97、A/Fujian/411/2002处于不同的基因进化分枝上,时间间隔越久,进化距离越远,同样的结果也出现在抗原变异分析中.而H1N1分离株的基因变异情况和抗原变异情况则不一致,在基因变异中,与疫苗推荐株A/New Caledonia/20/1999的距离远近受到分离时间的影响,抗原变异还与病毒是否从散发病例或聚集性病例分离有关.结论 流感病毒血凝素的基因变异和抗原变异的结果基本吻合,采用流行株免疫血清的血凝抑制试验能更好地反映病毒的变异和进化情况.     

生物巩怖主义的威胁及防御策略

近期美国遭遇炭疽侵袭 ,引发恐慌 ,恐怖组织欲利用生物武器 ,由来已久。目前有关生物性攻击的威胁 ,生物恐怖的概念以及可能造成的灾难和人类将面临何种生物威胁都成为关注的焦点。生物恐怖活动的可能后果是什么 ?1999年 2月 ,在美国弗吉尼亚州阿灵顿市召开了首届医疗公共卫生防御生物恐怖活动对策研讨会。此次大会共有公共卫生职员、内科医生和其它医学团体以及政府官员、军事人员和情报专家等 95 0名参加 ,分别来自美国 4 6个州、华盛顿市、澳大利亚、奥地利、加拿大、英国、芬兰、法国、德国、以色列、意大利、荷兰等不同的地区和国家。…     

甲型H1N1流行性感冒病毒血凝素蛋白基因进化研究

Objective To determine the evolutionary rate and divergence time of influenza A virus HA gene isolated recently worldwide pandemic and explore the origin and its transmission. Methods A total of 344 HI sequences available in the GenBank (including 248 isolated from human, 84 from swine, 11 from avian, and 1 from ferret) and 7 isolated in Shanghai were collected. The nucleotide substitution rate and time to most recent common ancestor (tMRCA) was calculated using molecular clock theory and Bayesian Skyline Plot (BSP) based on Markov chain Monte Carlo. Then genetic phylogeny was constructed referring to posterior distribution. Results It was found that H1 sequences in the US from human, swine and avian were clustered significantly with swine H1 ones from Asia phylogenetieally (Cluster US). The second cluster (Cluster Eurasian Human) nearly consisted of human H1 sequences isolated in other regions. The third cluster (Cluster Eurasian Animal) consisted of swine and avian H1 sequences from China and Italy respectively. As for all the H1 sequences, the evolutionary rate was of 2.57×10-3substitutions/site per year averagely (95% Highest Posterior Density: 1.96×10-3-3.03×10-3/site per year). The estimated dates for tMRCA of human H1 in Europe and swine H1 in the mainland of China were the earliest, with the corresponding rates of 6.46×10-3/site per year and 0.97×10-3/site per year respectively. The tMRCAs of human and swine H1 sequences from the US were similar, with the rates of 5.86×10-3/site per year and 5.02×10-3/site per year. Conclusion The present flu outbreak was possibly induced by long-term circulation of influenza A virus (H1 N1) in human population and swine herds in America. There was no evidence proving that influenza virus in China involved in the present outbreak.     

无锡地区流感病毒病原学监测及H3亚型血凝素基因变异研究

目的 监测无锡地区2005年至2008年季节性流感型与亚型分布并了解2008年部分A/H3N2分离株血凝素(8A)基因变异状况.方法 无锡地区医院门诊流感样患者及集体单位聚集性流感样暴发患者采集鼻咽拭标本,接种MDCK细胞,采用标准抗血清鉴定阳性分离株型与亚型,并对2008年部分H3亚型流感病毒进行HA全基因测序,分析HA基因变异状况.结果 2005年至2008年9月,在无锡地区流感样患者中共分离到435株流感病毒,其中164株为A/H1N1亚型,80株为MH3N2亚型,B型191株.型与亚型有明显季节性分布强弱特征.H3亚型HA序列分析,无锡地区9株分离株与上海地区同期分离株接近,有很多序列相互穿插归属于进化树的同一分支,与WHO 2008-2009年疫苗推荐株相近.结论 近年无锡地区散发和局部暴发流感病毒感染仍主要为A/H1N1、A/H3N2和B型,A/H3N2 HA基因与上海地区同期分离株接近,与WHO 2008-2009年疫苗推荐株相近.     

无锡地区流感病毒病原学监测及H3亚型血凝素基因变异研究

目的 监测无锡地区2005年至2008年季节性流感型与亚型分布并了解2008年部分A/H3N2分离株血凝素(8A)基因变异状况.方法 无锡地区医院门诊流感样患者及集体单位聚集性流感样暴发患者采集鼻咽拭标本,接种MDCK细胞,采用标准抗血清鉴定阳性分离株型与亚型,并对2008年部分H3亚型流感病毒进行HA全基因测序,分析HA基因变异状况.结果 2005年至2008年9月,在无锡地区流感样患者中共分离到435株流感病毒,其中164株为A/H1N1亚型,80株为MH3N2亚型,B型191株.型与亚型有明显季节性分布强弱特征.H3亚型HA序列分析,无锡地区9株分离株与上海地区同期分离株接近,有很多序列相互穿插归属于进化树的同一分支,与WHO 2008-2009年疫苗推荐株相近.结论 近年无锡地区散发和局部暴发流感病毒感染仍主要为A/H1N1、A/H3N2和B型,A/H3N2 HA基因与上海地区同期分离株接近,与WHO 2008-2009年疫苗推荐株相近.     

甲型H1N1流行性感冒病毒血凝素蛋白基因进化研究

Objective To determine the evolutionary rate and divergence time of influenza A virus HA gene isolated recently worldwide pandemic and explore the origin and its transmission. Methods A total of 344 HI sequences available in the GenBank (including 248 isolated from human, 84 from swine, 11 from avian, and 1 from ferret) and 7 isolated in Shanghai were collected. The nucleotide substitution rate and time to most recent common ancestor (tMRCA) was calculated using molecular clock theory and Bayesian Skyline Plot (BSP) based on Markov chain Monte Carlo. Then genetic phylogeny was constructed referring to posterior distribution. Results It was found that H1 sequences in the US from human, swine and avian were clustered significantly with swine H1 ones from Asia phylogenetieally (Cluster US). The second cluster (Cluster Eurasian Human) nearly consisted of human H1 sequences isolated in other regions. The third cluster (Cluster Eurasian Animal) consisted of swine and avian H1 sequences from China and Italy respectively. As for all the H1 sequences, the evolutionary rate was of 2.57×10-3substitutions/site per year averagely (95% Highest Posterior Density: 1.96×10-3-3.03×10-3/site per year). The estimated dates for tMRCA of human H1 in Europe and swine H1 in the mainland of China were the earliest, with the corresponding rates of 6.46×10-3/site per year and 0.97×10-3/site per year respectively. The tMRCAs of human and swine H1 sequences from the US were similar, with the rates of 5.86×10-3/site per year and 5.02×10-3/site per year. Conclusion The present flu outbreak was possibly induced by long-term circulation of influenza A virus (H1 N1) in human population and swine herds in America. There was no evidence proving that influenza virus in China involved in the present outbreak.     

上海地区水痘带状疱疹病毒基因型研究

目的：了解上海地区水痘-带状疱疹病毒基因型。方法：用Vero和Vero-E6细胞分离培养临床诊断水痘病人的咽拭子标本,观察病毒的致细胞病变效应（CPE）,对病变细胞培养液提取DNA,进行聚合酶链反应（PCR）,对PCR产物测序,将测序结果与Genbank中公布的水痘病毒序列比对判定水痘带状疱疹病毒和病毒的基因型。结果：PCR产物进行电泳,在550 bp处可见目的片段。测序结果证实为水痘带状疱疹病毒,基因型为J型。结论：从2006年5月华山医院临床诊断水痘患者咽拭子标本分离到的水痘带状疱疹病毒基因型为J型,ORF22读码框与水痘带状疱疹减毒活疫苗V-Oka株基因同源。