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RT-PCR检测犬粪便中的冠状病毒 总被引：1，自引：2，他引：1
根据犬冠状病毒(caninecoronavirus, CCV) 的S基因序列, 用计算机设计并合成了1对引物P1、P2, 以此引物及以从美国进口的CCV疫苗的反转录产物为模板, 初步建立了检测CCV的RT PCR。将纯化的PCR产物成功地克隆到pGEM T easy载体中, 鉴定、测序并进行同源性分析。结果表明, 与1 71和UCD 1株同源性分别为91 5%和94 3%。该RT PCR能对疫苗中CCV及CCV参考株USCV B1的S基因进行特异性地扩增,长度为575bp, 对犬的其他病毒及CRFK细胞未能扩增出任何片段, 该RT PCR能检测出0 5μLCCV培养液/2g粪便。用建立的RT PCR在上海对50条犬粪便进行检测, 结果表明CCV阳性6例。本研究建立了检测犬粪便中的CCV的RT PCR, 能用于犬冠状病毒流行病学调查。 相似文献
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大熊猫犬冠状病毒的分离与鉴定 总被引：11，自引：2，他引：11
以犬肾传代细胞(MDCK)从1只病死大熊猫肝脏中分离到1株病毒,命名为DXMV。电镜观察磷钨酸负染的病毒细胞培养物，发现冠状病毒样粒子，超薄切片上可见细胞浆内病毒包涵体。理化学和生物学鉴定结果表明，DXMV核酸类型为RNA，对氯仿、乙醚敏感，可耐受1％胰蛋白酶的作用，具有一定的耐酸性和耐热性，可在MDCK、FCWF、F81细胞中增殖，无血凝性和血吸附特性。血清中和试验结果表明，DXMV可被犬冠状病毒阳性血清中和。采用犬冠状病毒间接荧光抗体法染色DXMV细胞飞片，可在细胞浆内见到特异性荧光。以冠状病毒通用引物和犬冠状病毒特异性引物，可从大熊猫肝脏和不同代次的病毒细胞培养物中扩增出与预期值251bp和515bp大小相同的核苷酸片段。由此说明，该病毒为犬冠状病毒，大熊猫可被犬冠状病毒感染。 相似文献
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Two pairs of PCR primers were designed according to sequence of canine distemper virus （CDV） and canine coronavirus （CCV） from GenBank, respectively, which could amplify 550 bp fragment for CDV and 225 bp fragment for CCV. The products of PCR were cloned to pMD18-T for sequencing, which proved to be specific. Positive plasma were developed for standard DNA, and the sensitivity result of duplex PCR showed that the method could amplify 0.1 ng/μL nucleic acid for both of viruses. The result of rudimentary application showed that the method was specific, sensitive, efficient, and was a new detecting method for the mixed infection of CDV and CCV. 相似文献
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动物冠状病毒通用PCR方法的建立及基因序列分析 总被引：3，自引：0，他引：3
参考GenBank中公布的冠状病毒相关序列,根据动物冠状病毒聚合酶基因的保守区段设计一对通用引物。用这对引物对犬冠状病毒、猫冠状病毒等8种动物冠状病毒的cDNA模板进行通用PCR扩增和通用PCR反应条件的优化,结果均得到与试验设计相符的449 bp条带;特异性试验结果表明犬冠状病毒、猫冠状病毒等均扩增出449 bp目的条带,而阴性对照没有。敏感性试验结果表明,其敏感程度与常规PCR方法相同。将扩增的聚合酶基因与冠状病毒的参考毒株进行同源性比对,同源率在56.69%～99.55%之间。进化树分析结果与文献中对冠状病毒根据血清学与基因学角度分类报道相一致。 相似文献
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Polymerase chain reaction was used to detect an economically important herpesvirus, channel catfish virus (CCV). A segment of the viral DNA was sequenced and oligonucleotide primers were produced from that sequence. After the primers were tested for the possibility of hybridization to catfish DNA, they were used to prime the polymerase chain reaction, using pure CCV DNA, CCV DNA added to catfish DNA, and DNA from catfish infected and not infected with CCV. In all cases, the method proved to be simple and sensitive in its detection by CCV DNA. When catfish DNA was present, less than 0.1 pg of CCV DNA was detectable. Channel catfish virus DNA in a latent carrier of CCV was readily detectable. 相似文献
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Seok-Young Jeoung So-Yun Ann Hyun-Tae Kim Doo Kim 《Journal of veterinary science (Suw?n-si, Korea)》2014,15(4):495-502
The purpose of this study was to investigate the genetic features of canine coronavirus (CCV) strains detected in Korea. M gene sequences obtained for isolates from 22 dogs with enteritis over a 5-year period were evaluated. Sequence comparison revealed that the 22 Korean CCV strains had an 87.2 to 100% nucleotide homology. Comparing to the typical reference CCV strains (type II), the nucleotide sequence of Korean strains had homology ranged from 86.3% to 98.3% (89.1% to 99.2% for the amino acid sequence) and 87.7% to 97.8% (92.4% to 100% for the amino acid sequence) when compared to FCoV-like CCV strains (type I). Three amino acid variations in the M gene were characteristic for the Korean CCV strains. Phylogenetic analysis demonstrated that the 22 Korean CCV strains belonged to four typical CCV clusters (i.e., a unique Korean CCV cluster, a type II and transmissible gastroenteritis virus cluster, an intermediate cluster between type I and II, and a type I cluster). This study was the first to identify genetic differences of the M gene from Korean CCV strains and provided a platform for molecular identification of different Korean CCV strains. 相似文献
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Restriction endonuclease patterns of some European and American isolates of avian infectious laryngotracheitis virus 总被引：2，自引：0，他引：2
Eleven isolates of infectious laryngotracheitis virus (nine European and two American) were compared by restriction endonuclease analysis of their DNA after radiolabeling with 32P. Digestion with KpnI gave identical cleavage patterns for all the European isolates, but the two American viruses (one field and one vaccine) showed some differences from them and from each other. In the case of the American vaccine strain, however, these differences were only minor. After BamHI digestion, only the American field isolate appeared to be different, whereas with HindIII, all 11 isolates were identical. 相似文献
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Six modified-live (ML) infectious laryngotracheitis (ILT) vaccine viruses, three reference strains, and 18 field isolates were compared by restriction endonuclease analysis of their DNA. Viral DNA digestion patterns were established for vaccine viruses using restriction endonucleases PstI, BamHI, KpnI, and HindIII. Using these enzymes, five of six ML vaccine viruses had identical restriction endonuclease cleavage patterns. Vaccine viruses had distinct patterns compared with ILT virus reference strains Illinois-N71851, Cover, and NVSL. Restriction endonuclease cleavage patterns of 18 field isolates of ILT virus, obtained from ILT outbreaks in North Carolina, were indistinguishable from vaccine viruses. These results suggest a possible role of vaccine or vaccine-like viruses in recent ILT outbreaks. 相似文献
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采用蔗糖密度梯度离心,纯化浓缩犬冠状病毒(CCV)、猫冠状病毒(FCV)、猫传染性腹膜炎病毒(FIPV)、猪传染性胃肠炎病毒(TGEV)、猪呼吸道冠状病毒(PRCV)的细胞培养物,分别设计7,17,11,10和4对引物,构建了49个基因片段的克隆。煮沸裂解法制备质粒DNA,回收PCR扩增产物,点制冠状病毒基因芯片。抽提病毒总RNA,利用Cy3-dCTP随机渗入反转录PCR标记,与芯片进行杂交检测,淘汰交叉的克隆片段。结果表明:克隆CCV1,CCV2,CCV5和CCV7可特异诊断CCV,克隆FCV6,FCV7,FCV8和FCV9可特异诊断FCV,克隆FIPV2,FIPV7,FIPV8和FIPV9可特异诊断FIPV,克隆PRCV1,PRCV2和PRCV3可特异诊断PRCV,克隆TGEV3,TGEV4,TGEV5和TGEV6可特异诊断TGEV。将这些特异克隆扩增片段重新点制基因芯片,与病毒PCR产物杂交,未发现交叉现象。基因芯片检测比传统PCR敏感1000倍,可有效应用于这5种动物冠状病毒的检测与区分。 相似文献
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利用PCR方法成功克隆了鸡痘病毒（Fowl poxvirus，FPV）4b核心蛋白基因549bp片段，序列分析表明，该序列与模板DNA（AF198100）碱基序列的同源性为99．45％，只有3个碱基差异，第215位由C→T，第386位由T→A，第388位由G→A。回收FPV4b核心蛋白基因549bp片段，以其为模板制备了地高辛标记的DNA探针。对新标记的探针进行标记效率检测，结果显示其标记效率为100mg／L；敏感性检测表明，该探针对同源DNA的检出限量为10Pg；特异性检测结果表明，用本试验所标记的探针对提取的FPV282E4和FPV儿株DNA、重组质粒pMD 18-T-4b进行检测结果均呈阳性，而鸡马立克氏病病毒、鸡传染性喉气管炎病毒、CEF的核酸提取物均成阴性，说明该探针具有较强的特异性。初步应用表明，本试验所建立的FPV的基因探针检测法可用于FPV的检测。 相似文献
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从规模化养殖场鸭群气管和泄殖腔试子分离到新城疫病毒(NDV)27株,用2株针对NDV HN单抗进行抗原表位分析,并选择4个分离株进行F基因高变区(374bp)和HN基因全长序列分析。抗原表位分析结果显示,27个鸭分离株均能与其中一株单抗C3-B7反应,而与另外一株单抗1E5反应为阴性。F基因(374bp)序列分析结果显示,4个鸭分离株均属于NDV ClassⅠ分支,分离株之间核苷酸同源性为99.2%～100%;分离株与NDV ClassⅡ毒株遗传距离为0.9%～9.9%,与NDV ClassⅠ毒株遗传距离为38.5%～41.7%。根据核苷酸序列推导的氨基酸序列表明,4个鸭NDV分离株F蛋白裂解位点氨基酸模式为:112-EROERL-117。HN基因序列分析结果显示,4个鸭NDV分离株HN基因全长1851bp,编码585个氨基酸;同源性比较发现4个鸭NDV分离株之间核苷酸同源性为99.7%～99.8%,与NDV ClassⅡ毒株核苷酸同源性为68.4%～70.5%,与NDV ClassⅠ毒株核苷酸同源性为95.8%～98.0%。本研究结果显示,鸭分离毒均属于NDV ClassⅠ弱毒,在抗原表位和基因序列上与广泛应用的NDV弱毒疫苗株(LaSota)不同,这些毒株的来源有待进一步深入研究。 相似文献
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Saber Jelokhani-Niaraki Majid Esmaelizad Morteza Daliri Rasoul Vaez-Torshizi Morteza Kamalzadeh Mohsen Lotfi 《Journal of veterinary science (Suw?n-si, Korea)》2010,11(3):243-247
The A Iran 05 foot-and-mouth disease virus (FMDV) subtype was detected in Iran during 2005 and has proven to be highly virulent. This study was undertaken to focus on molecular and phylogenetic analysis of 3A and 3B coding-regions in the A Iran 05 field isolate. To assess the genetic relatedness of A Iran 05 isolate the nucleotide and predicted amino acid sequences of the 3AB region of type A FMDV isolates were compared with twenty previously described type A FMDV isolates. The phylogenetic tree based on the 672 bp 3AB gene sequences of type A FMDV from thirteen different locations clustered them into five distinct lineages. The A Iran 05 isolate clustered in lineage A along with four type A variants and was closely matched with viruses isolated in Turkey and Pakistan during 2005~2006. The number of protein sequence differences exhibited by each of the isolates revealed that A Iran 05 isolate contains three amino acid substitutions at positions 47 and 119 of 3A and 27 of the 3B coding region. The nucleotide identity between A Iran 05 and the other four isolates of lineage A was estimated to be 98%. 相似文献
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Very virulent infectious bursal disease viruses (vvIBDVs) were detected in phenol inactivated bursal samples obtained from Brazil, the Dominican Republic, and Venezuela. After nucleotide sequence analysis of the hypervariable region of VP2 gene, the vvIBDVs from Brazil and Venezuela exhibited all of the 14 nucleotide changes that are conserved in the European UK-661 and most other vvIBDV strains. However, the vvIBDV from the Dominican Republic presented 11 nucleotide changes that are conserved in vvIBDV strains. After phylogenetic analysis, the Latin American strains were found to be related to other vvIBDV strains from Europe, Asia, and Africa. However, Brazilian and Dominican vvIBDVs clustered in two separate subgroups, while the vvIBDVs from Venezuela were closely related to other strains from other parts of the world. By deduced amino acid sequence, the three conserved amino acid residues in vvIBDV strains (222 Ala, 256 Ile, and 294 Ile) were confirmed in the Latin American viruses, and one amino acid change (300 Ala) was unique to all vvIBDVs from the Dominican Republic. The occurrence of this change in the Dominican vvIBDVs may have an impact in their antigenic makeup. Results of this study indicate that the vvIBDVs detected in Latin America are genetically similar to IBDV strains from other parts of the world. However, vvIBDVs from Venezuela were more similar to the vvIBDV strains from Europe and Asia. Of all the samples analyzed, vvIBDVs from Brazil and the Dominican Republic exhibited more genetic changes. These changes may have emerged as a result of the different management practices and environmental conditions present in each particular geographic area. 相似文献
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犬冠状病毒核酸探针的制备及其基因序列的测定与比较 总被引：1，自引：0，他引：1
采用RT-PCR方法扩增犬冠状病毒(CCV)YS1、C11和NL-18株5′端部分S基因序列,以随机插入DNA法对CCV YS1株纯化的PCR产物标记32P同位素,制备核酸探针,并与3株CCV反转录产物杂交.用平端连接法将3株CCV PCR产物克隆于pUC19 Sma I位点或pGEM-T载体中,经PCR鉴定为正确重组质粒.以双脱氧末端终止法测定了重组质粒的cDNA核苷酸序列,并用DNASIS计算机软件进行多重比较分析,绘制系统树.结果表明,所制备的核酸探针可与3株CCV反转录产物杂交,其核苷酸序列与多株CCV相应序列的同源性高达91.9%～99.1%,为CCV的保守区.由此说明,制备的核酸探针可用于犬CCV感染的分子流行病学研究. 相似文献
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Eight field isolates of bovid herpesvirus type 4 (BHV-4) were examined by restriction analysis and Southern blot hybridization with respect to their relatedness to one another and to the BHV-4 prototype strain DN-599. Isolates were obtained from cattle exhibiting a range of disease states including abortion, pneumonia, enteritis, metritis, and vaginal blisters. Initial growth studies of all 9 viruses were performed and revealed that the overall rate of virus growth was slow when compared with that of other herpesviruses. Infection with each virus also resulted in the formation of large fused cells, which in addition to the slow growth rate, indicated that the isolates were of the cytomegalovirus type. Further studies to characterize and compare the various BHV-4 isolates were undertaken by obtaining cell-free virus from infected cell populations. Viral isolates were purified and used as a source of BHV-4 DNA. Purified DNA, representing each of the 8 field isolates and the prototype strain DN-599, were each cleaved with 3 restriction enzymes and were separated by agarose-gel electrophoresis, and the resultant fragment patterns were compared. In general, genomic fragments of the field isolates corresponded to those generated by cleavage of DN-599 DNA, with the exception of the abortion-associated isolate 83-3572. Additional minor differences were also seen between DN-599 DNA and DNA from the other field isolates, but the overall restriction patterns were similar. To confirm that all isolates were members of the BHV-4 type, hybridization studies were performed using DN-599.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献