B组轮状病毒的分子生物学研究进展

除Ａ组轮状病毒外，引起人类腹泻的还有Ｂ组和Ｃ组轮状病毒。本文就Ｂ组轮状病毒的基因结构、功能、所编码的蛋白及其与Ａ组轮状病毒蛋白的对应关系以及Ａ、Ｂ、Ｃ组间和组内不同株轮状病毒主要基因片段之间的遗传同源性关系作一概述。     

C组轮状病毒的分子生物学研究进展

通过基因组的ｃＤＮＡ克隆序列测定和与Ａ组轮状病毒的比较分析,Ｃ组轮状病毒部分蛋白的编码基因已确定。现有资料表明在引起人类腹泻的Ａ、Ｂ、Ｃ三组轮状病毒中,Ａ组与Ｃ组的遗传学关系更为密切,不同来源的Ｃ组轮状病毒株之间存在高度的序列同源性。     

在昆虫细胞中表达G2型轮状病毒地方株VP7基因

Ａ组轮状病毒是导致婴幼儿重症腹泻的最主要病毒病原,ＶＰ７是病毒外壳上的主要糖蛋白,它具有中和抗原活性,与病毒的毒力及免疫保护性有关,也是划分病毒血清型的最主要标志之一〔１〕。ＶＰ７基因及其编码蛋白一直是人们研究的主要对象,很多研究工作和诊断试剂都需大...     

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...     

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。     

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。     

婴幼儿轮状病毒肠炎48例病原学检测及治疗

轮状病毒是婴幼儿秋冬季腹泻的最常见的病原。我们用ＥＬＩＳＡ试剂盒对５４例大便常规检查无ＷＢＣ或偶见的疑似轮状病毒腹泻患儿进行了轮状病毒病原学检测,从中检出轮状病毒阳性患儿４８例,被确诊为轮状病毒肠炎。结果显示粪便中轮状病毒滴度越高,其临床症状也相应越重。此４８例患儿均采用病毒唑、双歧杆菌活菌制剂、补液纠正脱水等综合治疗,其中３０例辅以消化道粘膜保护剂思密达治疗,治疗组显效率为６０％,而对照组为１１．１％,经ｔ检验两组间显效率及总有效率均有非常显著性差异。表明思密达用于轮状病毒肠炎的治疗是一种较为有效的方法。     

G5型人A组轮状病毒LL36755株的VP4、VP6和NSP4编码基因的分子特征

最近在亚洲首次发现并报道了感染人的G5型人A组轮状病毒LL36755株,为进一步探讨其进化来源,克隆了G5型人A组轮状病毒LL36755株的VP4、VP6、NSP4编码基因,并分析其基因序列的分子特征。结果发现卢龙株LL36755为罕见的G5P[6]型,其VP6的亚群为SGⅡ型,NSP4的基因型为B型。系统进化树分析表明,卢龙株LL36755的VP7、VP4编码基因与猪来源的毒株关系密切,而VP6、NSP4编码基因与人来源的毒株紧密相联系。可以推断新的人腹泻A组轮状病毒LL36755株是猪的VP7,VP4编码基因与人的VP6,NSP4编码基因的自然重组;而且该毒株不是G5的原型,很可能是人类轮状病毒与猪轮状病毒毒株的自然重组后逐步进化而来。     

人轮状病毒感染昆明小鼠乳鼠模型的建立

目的建立人轮状病毒G3型709株感染4d龄昆明小鼠乳鼠模型。方法通过灌胃病毒的方式造模,观察乳鼠被病毒攻击后不同时间其临床表现、小肠组织病理改变、小肠组织上皮细胞超微结构改变。酶联免疫吸附法检测轮状病毒抗原在乳鼠粪便中的表达,免疫荧光法检测轮状病毒在乳鼠小肠组织中的表达。结果4d龄昆明小鼠乳鼠被轮状病毒攻击24h后出现腹泻表现和小肠组织病理改变,72h最严重,之后腹泻率下降,病理改变减轻,第7天腹泻停止,病理改变消失。乳鼠小肠上皮细胞出现糖、脂肪代谢紊乱,其粪便和小肠组织中都可以检测出轮状病毒抗原表达。结论4d龄昆明小鼠乳鼠被人轮状病毒A组G3型709株经口攻击后病毒能够在其体内复制,出现腹泻表现。该病毒感染腹泻过程具有自愈特点。     

抑癌基因nm23—H1在逆转录病毒载体的表达

癌症转移是癌症病人死亡的主要原因之一。抑制肿瘤转移的基因ｎｍ２３－Ｈ１与ｎｍ２３－Ｈ２分别编码二磷酸核苷激酶（ＮＤＰＫ）的Ａ与Ｂ亚基〔１〕,ｎｍ２３－Ｈ１基因能有效地抑制肿瘤转移〔２〕。ｎｍ２３－Ｈ１的表达水平与黑色素瘤〔２,３〕、乳腺癌〔４〕、卵巢...     

Detection and typing of Helicobacter pylori cagA/vacA genes by radioactive,one-step polymerase chain reaction in stool samples from children

The detection and molecular typing of Helicobacter pylori virulence genes in human stool specimens by polymerase chain reaction (PCR) require an adequate amount of bacterial DNA and an appropriately adjusted PCR protocol. DNA was isolated from stool samples of 39 H. pylori-infected and nine uninfected Colombian children using the QIAamp Kit following the manufacturer's instructions but with modifications. DNA templates were amplified for the vacA s and m regions and for the cagA gene by PCR using radioactively labeled (32P) primers. The modifications in the standard Qiagen protocol of stool DNA extraction increased the final concentration of eluted total stool DNA 4.7 times (117 +/- 17 versus 22 +/- 3 ng/microl; P < 0.0001). Nevertheless, its amplification by regular PCR programs (30-40 cycles) did not generate visible signals because of the very low ratio of H. pylori DNA to other DNA. PCR for 80 cycles successfully amplified vacA in 36/39 samples (sensitivity, 92.3%) and cagA fragments in 21/39 (53.8%) fecal DNA samples. Both s and m vacA regions were amplified in 33/36 (91.7%) DNA samples. The s1m1 genotype was the most commonly isolated variant, accounting for 17/36 or 47.2% of positive samples. The s2m2 genotype was ascertained to be frequent also (14/36 or 38.9%). Almost all (94.1%) s1m1 genotypes were cagA positive. The majority of s2m2 genotypes (78.6%) were not associated with the cagA gene. Neither cagA nor vacA fragments were amplified from DNA isolates of H. pylori-uninfected children nor from DNA isolated from six gastrointestinal bacterial strains (specificity, 100%). The data suggest that the proposed modified technique of DNA extraction and PCR assay of stool samples may be an effective and reliable noninvasive tool for the detection and typing of H. pylori cagA/vacA virulence genes in infected individuals.     

人B组轮状病毒vp7基因的克隆和序列分析

利用逆转录—聚合酶链反应(RT-PCR)技术,扩增了人B组轮状病毒WH—2vp7基因,连接到克隆载体pUCm—T,并对其基因序列进行了分析。WH—2与ADRV的同源性达98％,与印度加尔各达分离株CAL—1达92％,而与动物B组轮状病毒同源性差别较大,如与IDIR(鼠)同源性仅为58％,与WD653(牛)B组轮状病毒同源性为63％,与ATI(牛)同源性为61％。对vp7基因的二级结构分析发现其mRNA折叠形成多达18个发卡环状结构。VP7蛋白是249个氨基酸组成,分子：量为28．4kDa,含有3个潜在的N连接的糖基化位点和多个磷酰化位点,从氨基酸序列的同源性来看,WH—2与ADRV的同源性达99％,与CAL—1达95％,而IDIR仅为51％,说明了WH—2和ADRV的起源相同。此研究对了解B组轮状病毒基因的进化和变异规律具有重要意义,也将为B组轮状病毒预防性疫苗的研制提供科学的依据。     

不同DNA提取法对腺病毒和细小病毒PCR检测效果评估

分别利用硫氰酸胍(Guanidine thiocyanate,GuScN)抽提法、螯合树脂处理法和蛋白酶K-酚/氯仿抽提法从粪便样品中制备腺病毒DNA(dsDNA)或细小病毒DNA(ssDNA)然后进行PCR检测。结果显示硫氰酸胍抽提法、螯合树脂处理法能有效地去除粪便中影响PCR扩增的抑制物,提高PCR检测的敏感性,而传统蛋白酶K-酚/氯仿抽提法不能有效地去除PCR扩增的抑制物,影响PCR检测结果。在检测粪便中腺病毒时,硫氰酸胍、螯合树脂和蛋白酶-酚/氯仿抽提法分别允许检测5TCID_{50相似文献}   

Opisthorchis viverrini: detection by polymerase chain reaction (PCR) in human stool samples

A polymerase chain reaction (PCR) assay was evaluated for detection of Opisthorchis viverrini eggs in the stool specimens of light and heavily infected individuals in Khon Kaen province of Thailand. A total of 75 fecal specimens were analyzed by PCR following DNA extraction. All the microscopically positive samples were positive by PCR, while 23 of 30 (76.6%) microscopically negative samples were also PCR positive. The sensitivity of the assay was 5 eggs/g of stool. This method is potentially useful in the diagnosis of human opisthorchiasis in endemic areas for treatment and in epidemiological investigations.     

An alternative, sensitive method to detect Helicobacter pylori DNA in feces

Background: Despite the high sensitivity and specificity of PCR, detection of Helicobacter pylori DNA in feces is still challenging. Fecal samples contain inhibitory molecules that can prevent amplification of the target DNA. Even by using specific DNA extraction kits for stools, monitoring of infection by analyzing stool samples remains problematic and endorses the need for improved diagnostic methods. Materials and Methods: The newly proposed method uses selective hybridization of target DNA with biotin‐labeled probes, followed by DNA isolation with streptavidin‐coated magnetic beads. After three washing steps, the purified DNA can be amplified immediately using conventional or quantitative PCR. In order to test this technique on biological samples, Mongolian gerbils were infected with H. pylori ATCC 43504 and fecal samples were analyzed on days 1, 4, and 10 post infection. Results: A detection limit of one bacterial cell per 100 mg stool sample was established, but only after removal of the magnetic beads from the target DNA by heating. This resulted in a 10‐fold increase of sensitivity compared to a commercially available stool DNA extraction kit. Analysis of fecal samples from infected gerbils demonstrated the presence of H. pylori DNA on each time point, while the uninfected animal remained negative. Conclusions: The proposed technique allows detection of very low quantities of H. pylori DNA in biological samples. In laboratory animal models, detailed monitoring of infection and complete clearance of infection can be demonstrated thanks to the low detection limit.     

Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection

Background

In Schistosoma mansoni infection, diagnosis and control after treatment mainly rely on parasitological stool investigations which are laborious and have limited sensitivity. PCR methods have shown equal or superior sensitivity but preservation and storage methods limit their use in the field. Therefore, the use of occult blood detection cards (fecal cards) for easy sampling and storage of fecal samples for further PCR testing was evaluated in a pilot study.

Methodology

Stool specimens were collected in a highly endemic area for S. mansoni in Ethiopia and submitted in an investigator-blinded fashion to microscopic examination by Kato-Katz thick smear as well as to real-time PCR using either fresh frozen stool samples or stool smears on fecal cards which have been stored at ambient temperature for up to ten months.

Principal Findings

Out of 55 stool samples, 35 were positive by microscopy, 33 and 32 were positive by PCR of frozen samples and of fecal card samples, respectively. When microscopy was used as diagnostic “gold standard”, the sensitivity of PCR on fresh stool was 94.3% (95%-CI: 86.6; 100) and on fecal cards 91.4% (95%-CI: 82.2; 100).

Conclusions

The use of fecal cards proved to be a simple and useful method for stool collection and prolonged storage prior to PCR based diagnosis of S. mansoni infection. This technique may be a valuable approach for large scale surveillance and post treatment assessments     

Yersinia pseudotuberculosis infection in breeding monkeys: detection and analysis of strain diversity by PCR

In the last three decades, several monkeys reared in outdoor/indoor-outdoor breeding colonies and cages of the Primate Research Institute, Kyoto University, died of yersiniosis caused by Yersinia pseudotuberculosis, necessitating introduction of a method to detect the bacteria rapidly and thus allow preventive measures to be undertaken. A rapid nested polymerase chain reaction (PCR) method for identification of Y. pseudotuberculosis in fecal samples and a random amplified polymorphic DNA (RAPD)-PCR approach for distinguishing between bacterial strains were therefore developed. Yersinia pseudotuberculosis isolates from monkey specimens were found to be classifiable into several types. To determine the source of infection, hundreds of fecal samples of wild rats, pigeons, and sparrows were collected from around the breeding colonies and cages, and subjected to PCR analyses. Yersinia pseudotuberculosis was detected in 1.7% of the fecal samples of wild rats. The DNA fingerprints of the bacteria revealed by RAPD-PCR were the same as that of one strain isolated from macaques, suggesting the wild rat to be a possible source of infection.     

Identification of bacterial DNA markers for the detection of human fecal pollution in water

We used genome fragment enrichment and bioinformatics to identify several microbial DNA sequences with high potential for use as markers in PCR assays for detection of human fecal contamination in water. Following competitive solution-phase hybridization of total DNA from human and pig fecal samples, 351 plasmid clones were sequenced and were determined to define 289 different genomic DNA regions. These putative human-specific fecal bacterial DNA sequences were then analyzed by dot blot hybridization, which confirmed that 98% were present in the source human fecal microbial community and absent from the original pig fecal DNA extract. Comparative sequence analyses of these sequences suggested that a large number (43.5%) were predicted to encode bacterial secreted or surface-associated proteins. Deoxyoligonucleotide primers capable of annealing to a subset of 26 of the candidate sequences predicted to encode factors involved in interactions with host cells were then used in the PCR and did not amplify markers in DNA from any additional pig fecal specimens. These 26 PCR assays exhibited a range of specificity in tests with 11 other animal sources, with more than half amplifying markers only in specimens from dogs or cats. Four assays were more specific, detecting markers only in specimens from humans, including those from 18 different human populations examined. We then demonstrated the potential utility of these assays by using them to detect human fecal contamination in several impacted watersheds.     

Detection of PCR products of Escherichia coli O157:H7 in human stool samples using surface plasmon resonance (SPR)

A method for the rapid detection of verotoxin-producing Escherichia coli O157:H7 in stools was evaluated. Strains possessing Shiga toxin-2 (stx-2) genes were isolated from stool samples and amplified using oligonucleotide primers. Stools spiked with cultured E. coli O157:H7 (strain 298 or strain 1646) were detected to be polymerase chain reaction (PCR) positive at 10(2) cfu per 0.1 g of stool. Stool samples from patients and healthy carriers showed a high correlation between positive results for a PCR and the presence of verotoxin-producing E. coli O157:H7, confirmed by isolation of serotype O157:H7 on sorbitol MacConkey medium (10 of 10 stool samples). These PCR products could be detected using a BIAcore 2000 surface plasmon resonance device using peptide nucleic acid as a sensor probe. In this report we use this method for the rapid detection of DNA from significant pathogenic organisms.