限制性内切酶分析法对病毒感染性疾病诊断的意义

目的探讨限制性内切酶分析法在病毒感染性疾病诊断中的作用。方法通过对同批次疑似柯萨奇B组病毒感染的病毒性心肌炎及麻疹样发疹的患者血清分别采用RT-PCR法、限制性内切酶分析法进行检测,然后将其结果进行比较分析。结果限制性内切酶分析法的三次检测结果完全一致,较RT-PCR法的检测结果更精确。结论限制性内切酶分析法敏感性高,特异性强,是早期诊断柯萨奇B组病毒性疾病的最为精准的检测方法,并且为其它病毒感染性疾病的诊断提供了理论和实验依据。     

柯萨奇病毒B4 2B基因siRNA达载体的构建及其对柯萨奇病毒B4抑制作用的研究

目的构建针对柯萨奇病毒B4 2B基因的siRNA表达载体并检测该载体在体外培养细胞中对柯萨奇病毒B4的抑制作用.方法选择柯萨奇病毒B4的2B基因区21bp的基因片段作为靶序列,合成含有靶序列的DNA片段并克隆到pGCsi-U6/Neo/GFP/siNeGative载体中,构建pGCsi-U6/Neo/GFP/2B,使其可表达具有发夹结构的双链siR-NA,在转染该载体后用柯萨奇病毒B4攻击Hela细胞,检测该载体对病毒感染细胞的保护效应.结果通过酶切、电泳、序列分析及荧光显微镜观察证明载体构建成功,而且pGCsi-U6/Neo/GFP/2B转染组的柯萨奇病毒B4的滴度及TCID50都明显低于pGCsi-U6/Neo/GFP/siNeGative转染组.结论成功构建了柯萨奇病毒2B基因的siRNA表达载体,而且该载体在体外培养细胞中对柯萨奇病毒的复制具有抑制作用.     

柯萨奇病毒B4 2B基因siRNA表达载体的构建及其对柯萨奇病毒B4抑制作用的研究

目的构建针对柯萨奇病毒B42B基因的siRNA表达载体并检测该载体在体外培养细胞中对柯萨奇病毒B4的抑制作用。方法选择柯萨奇病毒B4的2B基因区21bp的基因片段作为靶序列，合成含有靶序列的DNA片段并克隆到pGCai-U6／Neo／GFP／siNeGative载体中，构建pGCsi-U6／Neo／GFP／2B，使其可表达具有发夹结构的双链siRNA，在转染该载体后用柯萨奇病毒B4攻击Hela细胞，检测该载体对病毒感染细胞的保护效应。结果通过酶切、电泳、序列分析及荧光显微镜观察证明载体构建成功，而且DGCsi-U6／Neo／GFP／2B转染组的柯萨奇病毒B4的滴度及TCID50都明显低于pGCsi-U6／Neo／GFP／siNeGative转染组。结论成功构建了柯萨奇病毒2B基因的siRNA表达载体，而且该载体在体外培养细胞中对柯萨奇病毒的复制具有抑制作用。     

酶扩增及核酸杂交对肠道病毒进行分子水平检测及鉴定

对肠道病毒基因组分析揭示,其5′—非转译区组具有高度保守性。作者利用此特性成功地设计了种属特异性引物,利用酶扩增技术从柯萨奇病毒B群(CVB)1～6型,脊髓灰质炎病毒(PV)1～3型,4种A 型柯萨奇病毒(CVA)和29种人肠道弧儿病毒(EV)扩增cDNA 证明,这种种属特异性引物不仅可用于肠道病毒的检测还可用于分类及鉴定。作者首先合成一组与5′—非转译区同源的引物E1、E2。该组引物可对6个CVB 血清型,所有3个PV 血清型,4个CVA 血清型和29个EV 血清型中的28个型别的cDNA 进行扩增。在所有41个检测的肠     

构建柯萨奇B4病毒非结构蛋白P2C与质粒PQE-4.0的重组质粒

背景:研究表明柯萨奇B4病毒感染与1型糖尿病发病有关,但目前尚未明确其关系的确切机制.目的:构建柯萨奇B4病毒非结构蛋白P2C与质粒PQE-4.0的重组质粒,转入大肠杆菌中表达,并对表达产物进行功能鉴定.设计、时间及地点:随机对照实验,于2007-03/08在吉林大学免疫学教研窒完成.材料:CVB4、HeLa细胞病毒均为吉林大学病原生物教研室保存;菌株E.coli DH5α与M15为东北师范大学遗传所保存;质粒PUCm-T载体、PQE4.0载体由东北师范大学曾宪录教授惠赠.方法:提取柯萨奇B4病毒总RNA,经反转录一聚合酶链反应扩增非结构蛋白P2C基因,与PUCm-T载体连接,将PUCm-T-P2C质粒进行BamH Ⅰ和HindⅢ双酶切,同收目的片断并定向克隆到PQE4.0表达载体中酶切鉴定.将PQE4.0-P2C 阳性重组质粒转化大肠杆菌M15,在IPTG诱导下表达.主要观察指标:①以十二烷基硫酸钠一聚丙烯酰胺凝胶电泳检测柯萨奇B4病毒非结构蛋白P2C的表达.②采用淋巴细胞转化实验和酶联免疫吸附实验检测大肠杆菌M15细胞重组蛋白粗提物.结果:①反转录-聚合酶链反应扩增得到的987 bp的cDNA片段,克隆入PUCm-T载体,酶切释放出长度约2773 bp和987 bp的两条片段,其中一条与非结构蛋白P2C的聚合酶链反应扩增产物片段大小基本一致,序列分析与Genbank 报道的序列一致.目的片段克隆入PQE4.0载体,酶切释放出长度约3 400 bp和987 bp的两条片段,其中一条与非结构蛋白P2C的聚合酶链反应扩增产物片段大小基本一致,获得PQE 4.0-P2C重组表达质粒.②十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示未诱导组无明显蚩白带,而诱导组在31 000位置均出现较明显的蛋白带,且随着诱导时间延长蛋白表达量也随之增加.③淋巴细胞转化实验与酶联接免疫吸附试验测定结果表明,表达产物具有良好的抗原性与特异性.结论:柯萨奇B4病毒非结构蛋白P2C在大肠杆菌中高效表达,且表达产物具有一定的免疫活性.     

柯萨奇B组病毒与小儿急性短暂性跛行发病关系的研究

目的探讨小儿下肢急性短暂性跛行发病的感染因素,为临床提供诊断依据。方法采用酶联免疫吸附试验对600例小儿急性跛行患儿组、100例上呼吸道感染组(下称上感组)、100例健康儿童对照组进行血清柯萨奇B组病毒1～6型(CVB 1～6型)IgM检测。结果小儿跛行患者组CVB-IgM阳性检出率为51.3%,其中以B1型为主,B4型次之,阳性率分别占36.8%和16.3%,检出双重阳性感染79例,占总阳性病例的25.7%(79/308);上感组CVB-IgM检出率为29.0%,型别亦以B1和B4为主,检出率依次为15.0%和10.0%,双重阳性占阳性病例的20.7%(6/29);健康对照组CVB-IgM检出率为16.0%,型别仍以B1和B4为主,检出率分别为6.0%和5.0%。3组的阳性率跛行组明显高于上感组和健康对照组,差异均有统计学意义(P<0.01)。结论柯萨奇B组病毒感染与小儿急性短暂性跛行发病关系密切,可能是其发病的重要感染因素之一,分型检测CVB特异性IgM抗体具有重要的临床诊断意义。     

荧光RT-PCR同步检测肠道病毒71型和柯萨奇病毒A组16型方法的建立及应用

目的建立快速、灵敏、特异的肠道病毒71型和柯萨奇病毒A16型的荧光PCR检测及分型方法。方法针对肠道病毒71型和柯萨奇病毒A16型的特异性保守基因vp1基因分别设计一对引物和对应的探针,对荧光RT-PCR反应体系进行优化,验证方法的特异性、灵敏度和重复性,并对临床样品进行检测分析。结果该方法对肠道病毒71型和柯萨奇病毒A16型的检测有高度特异性,与甲肝、腮腺炎、风疹、乙型脑炎等病毒均无交叉反应,检测灵敏度可达10拷贝/μl;43例患儿肛拭子标本中肠道病毒71型和柯萨奇病毒A16型检测率分别为37.21%(16/43)和13.95%(6/43)。结论荧光RT-PCR法分型检测肠道病毒71型和柯萨奇病毒A16型的方法具有快速、便捷、特异、灵敏等特点,适用于肠道病毒的早期诊断。     

柯萨奇病毒感染在I型糖尿病发病中的作用

Ⅰ型糖尿病(IDDM)的发病机制至今未明,一般认为与遗传、环境因素及病毒感染等有关。近期许多研究表明,Ⅰ型糖尿病属器官特异性自身免疫性疾病,在导致胰岛β细胞破坏的最初阶段,病毒感染是重要原因之一。Clements报告柯萨奇病毒感染可引起胰岛细胞白身免疫而致IDD。柯萨奇病毒为小RNA病毒科肠道病毒属的成员,分A、B两组。A组包括1～24型,B组包括1～6型。基因组为学单链正股RNA。我们对糖尿病患者进行了柯萨奇B_(1-6)特异性IgM检测,以探讨柯萨奇B组病毒感染在IDDM发病中的作用。     

柯萨奇病毒A组5型实时荧光RT-PCR检测方法的建立与评价

目的 建立柯萨奇病毒A组5型(CVA5)特异的一步法实时荧光反转录聚合酶链反应(RT-PCR)核酸检测方法。方法 以流行于遵义地区的CVA5病毒株和NCBI基因库中随机下载的CVA5 VP1基因序列作为参考序列,分析这些序列的保守区域,在其保守区设计特异性的引物与Taqman探针,建立检测CVA5的一步法实时荧光RT-PCR。通过构建含CVA5 VP1保守区域的重组质粒,绘制标准曲线,并对检测方法的灵敏度、特异性、重复性及临床样本检出限进行评价。利用该方法对遵义地区273份未明确分型的肠道病毒阳性样本进行检测,将检出的部分CVA5阳性产物进行测序。结果 成功建立一种基于实时荧光RT-PCR的CVA5检测方法,该方法在10⁴～10⁹copy/mL范围内具有良好的线性关系(R²>0.999),平均扩增效率为102.26%。灵敏度高,对临床样本的检出限为10³copy/mL。该方法的特异性强,与柯萨奇病毒A组2型(CVA2)、柯萨奇病毒A组4型(CVA4)、柯萨奇病毒A组6型(CVA6)、丙型肝炎病...     

p73β基因对肝癌细胞的影响

目的扩增及鉴定包含人类p73基因TAp73β转录本蛋白编码区的cDNA片段,将此片段克隆入真核表达载体,转染肝癌细胞,观察其对肝癌细胞生物活性的影响。方法从手术活体组织中提取总RNA,采用RT-PCR技术扩增TAp73β基因全长编码序列CDS（full-length coding sequence）,目的片段回收纯化后用限制性内切酶酶切及测序两种方法鉴定目的片段正确与否并检测其纯度、浓度;并转染肝癌Hep3B细胞,用MTT法进行细胞凋亡检测。结果该扩增片段经限制性核酸内切酶酶切图谱分析,与预期结果吻合,转染的肝癌Hep3B细胞出现凋亡。结论包含人类p73基因TAp73β转录本蛋白编码区的cDNA片段被成功扩增且末端带有限制性核酸内切酶XbaⅠ、KpnⅠ识别序列,它对肝癌细胞有抑制作用,从而为临床上肝癌的治疗提供了新思路。     

THE MAMMALIAN CELL-VIRUS RELATIONSHIP : VI. SUSTAINED INFECTION OF HELA CELLS BY COXSACKIE B3 VIRUS AND EFFECT ON SUPERINFECTION

Sustained infection of HeLa cells by Coxsackie B3 virus, dependent on presence of viral inhibitor in culture medium, was achieved. Persistent treatment of carrier cultures with anti-Coxsackie B3 hyperimmune monkey serum eventually eliminated virus from carrier cultures indicating that a lysogenic virus-cell relationship was not operative. Free virus was produced continuously by carrier cultures despite washing and neutralization with antiserum to eliminate free virus temporarily. In carrier cultures, about 1.5 to 1.9 plaque-forming units of virus per cell were cell-associated; approximately 6 per cent of this cell-associated virus was not neutralizable by antiserum. In growth medium containing anti-B3 antibody, cells from carrier cultures formed colonies as efficiently as cells from B3-cured cultures. Assays of carrier cultures for infectious centers indicated that less than 1 per cent of cells produced free infectious virus. The Coxsackie B3 virus-carrier state appeared to represent surface residence of B3 virus on the majority of carrier cells with restriction of productive infection to a small proportion of the population. Coxsackie B3 carrier HeLa cultures, unlike control cultures, were not destroyed by challenge with Coxsackie B1, B3, or B5 viruses. The B3 carrier state did not interfere with superinfection by herpes, vaccinia, and types 1 to 3 polioviruses. In contrast to parental or B3-cured lines, B3-carrier HeLa cultures superinfected with Coxsackie B1 virus produced no significant virus, and cultures superinfected with B5 viruses produced new virus to a limited extent only. Specific interference with Coxsackie virus superinfection by the B3-carrier state of HeLa cells was shown to be attributable to failure of attachment in the instance of Coxsackie B1 virus, and failure of penetration and/or eclipse in the instance of B5 virus. The interfering effect was circumvented successfully by superinfection of carrier cells with ribonucleic acid extracted from Coxsackie B1 and B5 viruses.     

急性心肌梗死与柯萨奇B组病毒感染关系的研究

目的　探讨柯萨奇B组病毒感染与急性心肌梗死 (AMI)的发病关系。方法　应用SPA吸收IgG结合间接ELISA法检测AMI患者血清中柯萨奇B组病毒特异性IgM抗体及中和抗体。结果　 4 8例AMI患者血清特异性IgM抗体阳性率为 75 % ,血清中和抗体阳性率为 85 4 % ,与正常对照组相比差异非常显著。双份血清试验 ,恢复期血清抗体效价高于初期血清抗体效价 4倍以上。结论　AMI发病与柯萨奇B组病毒感染有关。     

cDNA探针检测柯萨奇B_3病毒RNA

应用光敏生物素标记ｃＤＮＡ探针作打点杂交检测柯萨奇Ｂ＿３病毒（ＣＶＢ３）－ＲＮＡ，并采用薄层层析扫描仪反射扫描杂交信号和计算机整合反时峰面积的手段，对所测ＣＶＢ３－ＲＮＡ进行半定量。结果显示反射峰面积与进行自身杂交的ｃＤＮＡ含量（ｒ＝０．９８１，Ｐ＜０．００２５）及与病毒稀释度（ｒ＝０．９９７，Ｐ＜０．００５）呈有显著意义的直线正相关，提了薄层层析扫描结合核酸杂交的确可对ＣＶＢ３－ＲＮＡ进行半定量分析，也可为研究其他病毒或非病毒核酸提供新的检测方法，值得推广。     

Detection of enteroviruses using subgenomic probes of Coxsackie virus B4 by hybridization

The objective of this research was to develop group- and type-specific probes for the detection of enteroviruses. Coxsackie virus B4 RNA was cloned, and a series of subgenomic clones were generated. Six of these clones, containing sequences from the 3' end or the 5' end of the genome, were tested for their ability to detect these viruses in a small number of infected cells employing nucleic acid hybridization technique and total cytoplasmic RNA from a panel of 11 serotypes of enteroviruses. The RNA from cells infected with Coxsackie B viruses gave characteristic and positive hybridization signals. Coxsackie B-specific probes and a control Echo 9 probe detected Coxsackie A9 and Echo 3 weakly. As little as 0.5 microgram of the RNA--which contained 10-20 ng of poly(A)-containing, virus-specific, hybridizable RNA--was sufficient to successfully conduct the assay, suggesting high sensitivity of these probes. Probes that are 3' end-specific appear to be group specific, while those that are 5' end-specific appear to be type specific among the serotypes tested.     

病毒性肝炎患者中柯萨奇B组病毒的感染情况

目的：了解柯萨奇B组病毒（coxackie virus B,CVB)在病毒性肝炎中的感染情况。方法：用免疫酶法对203例各型肝炎组进行CVB1至6型免疫球蛋白G（IgG)检测，并与105例病毒性心肌炎组及正常对照组进行分析。结果：肝炎组阳性率（39．9％）高于正常对照组（13．4％），P＜0．005，而低于心肌炎组（53．3％），P＜0．025，B1－6型都有一定的检出率，且以各型交叉感染为主。结论：病毒性肝炎患者对CVB各型普遍易感，其致病性应引起重视与探讨。     

Genotyping of hepatitis B virus isolated from chronic hepatitis B patients in the south of Turkey by DNA cycle-sequencing method

The 8 genotypes of hepatitis B virus (HBV A-H) show a distinct geographic distribution and influence the course of disease and the prognosis of treatment. In this study, we have genotyped 50 HBV isolates circulating in the south of Turkey by DNA cycle sequencing, based on their compatibility with reference sequences of a part of S gene. In our cases, all 50 (100%) HBV sequences from the patients demonstrated full compatibility with the sequences of ayw subtype viruses in genotype D. However, we have found some nucleotide sequence variations within genotype D, 47 (94%) of which were related to HBVGEN1 (Z35716 genotype D) and 3 (6%) were related to HBVDNA (X68292, genotype D).     

应用PCR-RFLP技术检测阿德福韦耐药相关性HBV变异

目的建立一种简便、快速、实用的检测阿德福韦(ADV)耐药相关性HBV变异(rtA181V/T/S和rtN236T变异)的方法。方法根据GenBank收录的HBV基因全序设计套式-PCR引物,使野毒株的PCR产物上游末端引入BglI的酶切位点,使rt236变异株的PCR产物下游末端引入BseDI(SecI)的酶切位点,建立PCR-限制性片段长度多态性(RFLP)方法。用此方法分别检测含rt181变异株、rt236变异株及野毒株质粒和3例ADV耐药慢性乙型肝炎患者血清标本。将变异株和野毒株配成不同比例,再用此方法检测,以了解该方法的敏感性。结果本研究建立的PCR—RFLP方法可同时检测rt181变异和rt236变异;用此方法检测血清标本,结果与测序及克隆分析一致;此方法可检测出标本中10%的变异株。结论应用PCR- RFLP方法可同时检测rtA181V/T/S和rtN236T变异,具有较高的敏感性,可用于ADV耐药变异的早期诊断。     

Detection of influenza virus types A and B and type A subtypes (H1, H3, and H5) by multiplex polymerase chain reaction

Infections with influenza virus type A and B present serious public health problems on a global scale. However, only influenza A virus has been reported to cause fatal pandemic in many species. To provide suitable clinical management and prevent further virus transmission, efficient and effective clinical diagnosis is essential. Therefore, we developed multiplex PCR assays for detecting influenza types A and B and the subtypes of influenza A virus (H1, H3 and H5). Upon performing multiplex PCR assays with type-specific primer sets, the clearly distinguishable products representing influenza A and B virus were separated by agarose gel electrophoresis. In addition, the subtypes of influenza A virus (H1, H3 and H5), which are most common in humans, can be readily distinguished by PCR with subtype-specific primer sets, yielding PCR products of different sizes depending on which subtype has been amplified. This method was tested on 46 influenza virus positive specimens of avian and mammalian (dog and human) origins collected between 2006 and 2008. The sensitivity of this method, tested against known concentrations of each type and subtype specific plasmid, was established to detect 10(3) copies/microl. The method's specificity was determined by testing against other subtypes of influenza A virus (H2, H4 and H6-H15) and respiratory pathogens commonly found in humans. None of them could be amplified, thus excluding cross reactivity. In conclusion, the multiplex PCR assays developed are advantageous as to rapidity, specificity, and cost effectiveness.     

Virus infection and knee injury.

Serological evidence of virus infection was sought in 31 consecutive patients presenting with knee swelling and compared with age/sex-matched controls. In a normal age/sex-matched control group, 42% of patients had evidence of recent or past infection with Coxsackie B virus, emphasising the care required in the evaluation of the significance of Coxsackie B neutralization titres in individual patients. Of 12 patients presenting with knee swelling and a history of a twisting injury, eight had serological evidence of recent or past infection with Coxsackie B virus, and one had evidence of a current adenovirus infection.