两种PCR方法对检测A组轮状病毒膜芯片杂交结果的影响

A组轮状病毒(rotavirus,RV)是导致拿世界婴幼儿腹泻的最主要病原,危害巨大。拟用RT-巢式PCR技术对A组RV的保守序列进行高度扩增,通过固本室内制的膜芯片杂交,实现对该病毒的检测。分别采用对称PCR和不对称PCR扩增,均可得到扩增的目的片段．对称式扩增产物杂交结果不理想。而不对称式扩增得到了大量待检单链产物,同膜芯片杂交获得了理想的杂交结果。显著地提高了对A组RV杂交检测的灵敏度。表明不对称式PCR扩增是一种制备用于芯片杂交大量单链产物的理想方法,尤其是针对富含AT的核酸检测区域。     

膜芯片检测A组轮状病毒的初步研究

建立表面带正电荷的尼龙膜为基片的基因芯片,采用RT-semi-nested PCR方法,对A组轮状病毒RNA实现高度扩增,并通过5'端带地高辛标记的上游引物实现扩增产物的标记.通过同膜芯片上探针的杂交和免疫显色,实现对A组轮状病毒的检测.结果表明,膜芯片对探针的固定效果、杂交吸脱和检测结果可靠,空白对照和阴性对照均为阴性,探针均显示为阳性信号,并且信号强弱与探针浓度关系不大,而主要与探针本身同互补链的结合相关.以上结果说明已经初步建立了快速检测A组轮状病毒的膜基因芯片检测技术.     

从绵羊羔和山羊羔腹泻粪样中检出B组轮状病毒

用聚丙烯酰胺凝胶电泳法,从新疆巴里坤县的22份绵、山羊羔腹泻粪样中,检出11份具有B组轮状病毒的RNA电泳图型,联合电泳表明,绵羊羔与山羊羔的B组轮状病毒电泳图型完全相同,电镜观察表明,该病毒与典型轮状病毒(A组)具有相同的形态特征,但易降解破碎,口服接种剥夺初乳的绵羊羔和山羊羔,经11～13小时羔羊发生急性水样腹泻,并排出大量病毒,经A组轮状病毒组抗原ELISA检测,不具有共同组特异抗原,但经对流免疫电泳检测,它与成人腹泻轮状病毒(B组)具有共同组特异抗原,从而证实我国存在与国外报道相同的羊B组轮状病毒,既感染绵羊羔也感染山羊羔。是引起绵,山羊羔急性腹泻的不可忽视的病原之一。     

轮状病毒基因重配株血清型鉴定方法的研究

Ａ组轮状病毒中根据基因９的不同目前至少已发现有１３个不同Ｇ血清型,其中能引起人类致病的有Ｇ１－Ｇ４,Ｇ８,Ｇ９和Ｇ１２型。建立可靠的血清型鉴定技术对于轮状病毒疫苗的研制和分子流行病学的研究具有重要意义。本文首次报导了一种鉴定轮状病毒Ｇ血清型的新方法,利用已知有关轮状病毒ＶＰ７基因的序列资料,设计合成了一套鉴定轮状病毒Ｇ血清型的寡核苷酸探针,利用地高辛标记上述探针。待检品经反转录ＰＣＲ扩增后与上述一套寡核苷酸探针分别进行杂交得以确定其血清型。这一方法与目前常用的套式ＰＣＲ方法相比更适合于大量样品的操作而且结果可靠。用这一方法对本实验室组建的四株基因重配疫苗株进行实验,其结果与套式ＰＣＲ方法完全一致。     

健康儿童与轮状病毒感染儿童肠道菌群结构的比较研究

目的比较分析轮状病毒感染个体与健康个体肠道菌群结构的差异。方法采集11例轮状病毒感染个体及6例健康个体的粪便样品,提取粪便样品中细菌的混合DNA,先通过ERIC-PCR结合分子杂交的技术分析两组个体之间肠道微生物组成的相似性;再扩增粪便样品中菌群的16SrRNA基因,利用PCR—TGGE技术分析肠道菌群的组成情况。结果轮状病毒感染个体与健康个体相比,肠道菌群中GC含量较低的菌明显减少,同时肠道菌群有宿主专一性。结论轮状病毒感染会导致儿童肠道内菌群结构失调。     

在我国患急性腹泻成人中发现一种新轮状病毒

从3例急性成人腹泻患者粪便中,经电镜观察,成人腹泻轮状病毒—酶联免疫吸附试验(ADRV-ELISA)、普通轮状病毒—酶联免疫吸附试验(Rota-ELISA),猪抗C型(组)轮状病毒和鸡抗D型(组)轮状病毒血清分别与本病毒的免疫电镜试验以及RNA电泳等实验结果,发现了一种新轮状病毒,该病毒的形态结构与普通轮状病毒(Rotavirus)、成人腹泻论状病毒(Adult Diarrhoea Rotavirus,简称ADRV)极为相似,但抗原性与普通轮状病毒(A组),成人腹泻轮状病毒(B组),C型轮状病毒(C组),D型轮状病毒(D组)显然无关。基因分析表明,该病毒基因组由11个双链RNA片段组成,但其RNA电泳图型具有独自的特点,与目前公认的A组、B组、C组、D组论状病毒韵RNA电泳图型均不同,免疫电镜证实,该病毒能被人恢复期血清所凝集,表明该病毒可能是腹泻病人的病因。     

新成人腹泻轮状病毒J19株VP2、VP3的编码基因序列分析

利用一种改良的非依赖核酸序列的单引物扩增方法,从新成人腹泻轮状病毒J19株的核酸中扩增基因,克隆至pMD18-T中并进行测序和基因序列分析。J19株的VP2、VP3的编码基因为基因2、4,分别长2 969bp、2 204bp,它们分别编码973个氨基酸和719个氨基酸。J19株的VP2蛋白序列对B组人轮状病毒IDIR株的一致性为47.2%;J19株的VP3蛋白序列对C组人轮状病毒Cowden株一致性为25.1%。对J19株VP2的遗传进化分析表明,J19株在进化树上的位置靠近外群蛋白以及A、B和C组轮状病毒分枝的根部,并且它比较偏向于B组轮状病毒的分枝。这与VP6的遗传进化分析结果相一致。根据上述结果推测J19株可能是一个与B组轮状病毒的起源和进化密切相关的毒株之一;同时,这表明VP2在研究轮状病毒的遗传进化上具有重要价值。关于新成人腹泻轮状病毒J19株VP2、VP3的编码基因的序列分析,这是首次报道。     

健康婴幼儿粪便RV携带状况的调查研究

采用ELISA法对J60名6月至4周岁的健康婴幼儿粪便作了A组轮状病毒抗原检测,其中6月至2周岁婴幼儿阳性率为10％,3岁至4周岁儿童阳性率为6.9％,经统计学处理二组无显著差异(P>0.05),总阳性检出率为8.7％。表明A组轮状病毒在健康婴幼儿粪便中可以正常寄生。其年龄范同在6月至4周岁。     

长春地区轮状病毒流行株VP7基因的遗传变异

A组轮状病毒是引起婴幼儿秋冬季病毒性腹泻的主要病原.目前没有有效的治疗药物,应用安全而有效的疫苗是控制重症腹泻的首要措施.对当地A组轮状病毒流行株的主要中和抗原VP7的编码基因进行遗传变异分析,可以为疫苗的应用和开发提供有益的指导.利用ELISA方法对长春地区1999～2005年的腹泻患儿标本检测A组轮状病毒,RT-PCR方法对阳性标本进行G血清分型,发现长春地区2001年以后流行的轮状病毒以G3型血清为主.选取1999～2005年的G3型轮状病毒标本31份,对其VP7基因进行扩增、克隆、测序,经过计算机分析比对,31株G3型轮状病毒VP7基因核苷酸序列没有显著差异.同一流行季节的毒株具有较相似的遗传变异特征.在2003年轮状病毒流行季节内,有6株G3型分离株的VP7基因在碱基1 038位置上出现一个碱基缺失.毒株发生在A、B、C三个高变区的碱基突变,位点相同或者位置临近.2002年以后毒株的基因突变增加,非高变区的碱基变异增加,这可能有助于维持G3型轮状病毒成为流行株.有规律的变异多发生在高变区,但是非高变区的非连续性变异的增加值得引起注意.     

用反转录—聚合酶链反应检测B组轮状病毒

轮状病毒（Ｒｏｔａｖｉｒｕｓ）是属于呼肠病毒科（Ｒｅｏｖｉｒｉｄａｅ）的双链ＲＮＡ（ｄｓＲＮＡ）病毒。至今已将轮状病毒分为七个组（Ａ～Ｇ）。已经发现的Ｂ组轮状病毒分别来自人、大鼠、牛、猪、羊。近十年来,通过轮状病毒的研究,轮状病毒Ｂ组已被公认为引起人...     

婴幼儿腹泻A群轮状病毒G和P的基因分型研究

目的研究浙江萧山医院婴幼儿童腹泻标本中人轮状病毒（Human Rotavims）毒株的感染情况及G和P基因型流行特点。方法收集该院2009年8月至2010年8月腹泻儿童15233份粪便标本采用酶联免疫吸附试验、逆转录-巢式聚合酶链反应进行轮状病毒病原检测,将128份阳性标本进行VP7和VP4基本分型。结果15233份婴幼儿腹泻标本中有2706份标本为轮状病毒阳性,阳性率17．8％;男孩和女孩检出率差异无统计学意义,以6-12月龄段检出率最高;对128份阳性标本进行G血清分型和P基因分型,G1型53份（41．4％）、G3型38份（29．7％）、G1G3型17份（13．3％）、G未分型20份（15．6％）;P[8]型72份（56．3％）、P[4]型16份（12．5％）、P[8]P[4]型3份（2．3％）、P未分型37份（28．9％）,G血清型和P基因型的组合以G1P[8]为主,占29．7％（38／128）。结论浙江萧山医院A群轮状病毒G血清以G1型为主,其次为G3型,P基因型以P[8]型为主。     

Structure-function analysis of rotavirus NSP2 octamer by using a novel complementation system

Viral inclusion bodies, or viroplasms, that form in rotavirus-infected cells direct replication and packaging of the segmented double-stranded RNA (dsRNA) genome. NSP2, one of two rotavirus proteins needed for viroplasm assembly, possesses NTPase, RNA-binding, and helix-unwinding activities. NSP2 of the rotavirus group causing endemic infantile diarrhea (group A) was shown to self-assemble into large doughnut-shaped octamers with circumferential grooves and deep clefts containing nucleotide-binding histidine triad (HIT)-like motifs. Here, we demonstrate that NSP2 of group C rotavirus, a group that fails to reassort with group A viruses, retains the unique architecture of the group A octamer but differs in surface charge distribution. By using an NSP2-dependent complementation system, we show that the HIT-dependent NTPase activity of NSP2 is necessary for dsRNA synthesis, but not for viroplasm formation. The complementation system also showed that despite the retention of the octamer structure and the HIT-like fold, group C NSP2 failed to rescue replication and viroplasm formation in NSP2-deficient cells infected with group A rotavirus. The distinct differences in the surface charges on the Bristol and SA11 NSP2 octamers suggest that charge complementarity of the viroplasm-forming proteins guides the specificity of viroplasm formation and, possibly, reassortment restriction between rotavirus groups.     

大熊猫轮状病毒PCR检测方法的建立及应用

轮状病毒是威胁大熊猫健康的主要病原微生物之一。为了对大熊猫轮状病毒进行快速、方便且准确地检测,研发适合于基层饲养单位和保护区的检测方法是非常有必要的。本研究通过合成大熊猫轮状病毒Vp7基因序列,构建了PUC-VP7的重组质粒,并将其作为阳性对照对大熊猫轮状病毒样本进行PCR检测和分析。结果表明,在进行PCR扩增分析时,该质粒和病毒cDNA二者均在340 bp处出现了特异性条带。此外,对收集到的45份大熊猫粪便样本进行轮状病毒抗原检测时,其中2份样品在340 bp处出现条带,该基因片段与大熊猫轮状病毒CH-1株的相似性为99.89%。本研究构建的PUC-VP7质粒不但可以作为大熊猫轮状病毒PCR检测中的阳性质控品,而且还能有效地促进该PCR病毒检测技术在基层饲养单位和保护区的推广和应用。     

Use of PCR and nucleotide sequence analysis of the VP7 gene for detection of bovine rotaviruses and identification of their G serotypes

A Vp7 gene fragment PCR protocol was developed to detect the bovine rotaviruses and to identify their G serotypes. The most widespread bovine rotaviruses of G serotypes (G6, G8 and G10) can be distinguished on the basis of the PCR fragment size, while other G serotypes can be differentiated through a comparative analysis of the VP7 gene fragment nuclcotide sequence. Twenty-four bovine rotavirus field isolates were detected, and their G stereotypes were determined by using the method in question. Fourteen isolates were shown to be of G6 serotype; four of them were of G8, five--of G10, and one isolate was of G11 serotype. A possibility of detecting more than one isolate by this method was shown. Finally, a feasibility of using the method for searching for rotaviruses of new bovine rotavirus G serotypes and for rotaviruses, which do not belong to a so far described G serotypes, is discussed.     

Isolation of an atypical rotavirus causing diarrhea in neonatal ferrets

A rotavirus was isolated from neonatal ferrets (Mustela putorius furo) with diarrhea at a large commercial farm. This virus is classified as an atypical rotavirus, probably belonging to the group C rotaviruses. This classification is based on the lack of the rotavirus group A common antigen and on its distinct dsRNA electropherotype pattern in polyacrylamide gels. The diarrheal disease was reproduced experimentally in neonatal ferrets.     

Detection of Rotavirus from Hospitalized Diarrheic Children in Uttar Pradesh, India

In the present study 220 stool samples collected from diarrheic children admitted to different hospitals and nursing homes of Uttar Pradesh and Uttarakhand were screened for rotavirus. Of 220 diarrheic samples screened 46 samples were found to be positive for rotavirus by RNA PAGE. All the isolates exhibited 4-2-3-2 migration pattern suggesting group A rotavirus. Both long and short electropherotypes were prevalent in these regions. Six different electropherotypes were detected in this study period. Male diarrheic children were found to be more susceptible to rotavirus infection (22.96 %) than that of the female ones (17.64 %). Viral RNA isolated from stool samples again subjected to VP4 gene amplification by RT-PCR using con2 and con3 primer which resulted 876 bp product suggesting group A rotavirus. Besides virus isolation was successfully done using MA104 cell line.     

Characteristic of the etiologic structure of acute enteric infections in adults based on the data from infectious diseases hospital

Study of the etiologic spectrum of sporadic cases of causative agents of acute enteric infections (AEI) in 753 patients aged 15-85 years old using polymerase chain reaction (PCR) was performed in the infectious diseases hospital during 12 months. It was shown that detection of the causative agents of AEI by PCR was 2.2 times more effective than routine microbiologic diagnostics supplemented with test of feces on rotavirus by ELISA method and allowed to detect etiologic agent in 54.3% of the patients. Viral etiology of AEI was determined in 22.6% of the patients. The most significant causative agents of AEI comparable on the rate of detection with salmonella and shigella were genotype 2 noroviruses and group A rotaviruses which were isolated in 10.4 and 8.9% of the patients respectively. Seasonal change of dominating viral pathogens causing diarrheal diseases was noted: predominance of group A rotaviruses during winter-spring period, noroviruses--during spring-summer months, and astroviruses--during fall and in the beginning of winter.