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牛病毒性腹泻病毒RT-PCR检测方法的建立及应用 总被引：1，自引：1，他引：0
根据GenBank中登录的牛病毒性腹泻病毒(BVDV)基因序列,设计合成了1对特异性引物,建立了检测BVDV的RT-PCR方法。通过对该方法的特异性、敏感性和重复性进行试验,结果显示,该方法可从BVDV标准毒株Oregon C24V中扩增出471 bp的特异性片段,而对猪瘟病毒、牛传染性鼻气管炎病毒、牛呼吸道合胞体病毒、牛副流感病毒、MDBK正常细胞的扩增结果均为阴性。经对标准毒株的细胞毒进行检测,其敏感度达10-1 TCID50/mL。应用该方法对临床腹泻病牛各脏器样品进行检测,结果比病毒分离方法更为敏感,操作简便。表明建立的RT-PCR方法具有特异、灵敏、高效、快速的特点,可用于BVDV的临床检测及流行病学监测。 相似文献
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为了对疑似牛呼吸道合胞体病毒和巴氏杆菌混合感染的犊牛进行病原鉴定,本研究采用常规病毒经细菌分离鉴定和PCR方法分别进行分离与鉴定。结果表明,该病毒株能在BT细胞上增殖并产生特征性合胞体形态的细胞病变;无血凝性和血吸附特性;能被牛呼吸道合胞体病毒(bovine respiratory syncytial virus,BRSV)标准阳性血清中和;分离的病毒经RT-PCR鉴定为牛呼吸道合胞体病毒;根据菌落形态、细菌染色特性及生化特性,鉴定分离的细菌为巴氏杆菌。提示,该牛场为牛呼吸道合胞体病毒和巴氏杆菌混合感染。 相似文献
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为建立一种针对牛传染性鼻气管炎病毒(infectious bovine rhinortracheitis virus,IBRV)和牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)的双重PCR检测方法,根据GenBank中IBRV gB(UL27)和BVDV 5’-UTR基因序列分别合成特异性引物,优化反应条件,建立了能够同时检测IBRV和BVDV的双重PCR方法,并对其特异性、敏感性、重复性进行了测试。结果显示：该方法对IBRV、BVDV和IBRV/BVDV可分别扩增出500、198、500/198 bp的特异性目标条带,对牛呼吸道合胞体病毒、牛冠状病毒、牛细小病毒、牛纽布病毒和牛支原体检测结果均为阴性;对混合液中IBRV、BVDV的最低检测限分别为104和103 copies/μL;3次重复性试验结果一致。采用建立的方法对79份临床可疑牛血清样品进行IBRV、BVDV和IBRV+BVDV检测,阳性率分别为12.66%、17.72%和8.86%,与IBRV和BVDV单重PCR检测结果符合率分别为90.00%... 相似文献
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《中国预防兽医学报》2020,(3)
为建立以牛病毒性腹泻病毒(BVDV)非结构蛋白p80为包被抗原的间接ELISA方法,本研究将重组质粒pET30a-p80转化至大肠杆菌BL21(DE3)表达菌,将转化菌培养并诱导后,通过SDS-PAGE证实了p80蛋白以包涵体形式获得了表达,将纯化后的重组p80蛋白作为包被抗原,通过方阵滴定优化反应条件,建立了检测BVDV血清抗体的间接ELISA方法。特异性试验结果显示,该方法与BVDV阳性血清反应呈阳性,而与牛传染性鼻气管炎病毒、牛副流感病毒3型、牛呼吸道合胞体病毒、口蹄疫病毒及猪瘟病毒等常见病原的阳性血清无交叉反应,特异性较强。敏感性试验结果显示本实验建立的间接ELISA方法在阳性血清11 280倍稀释时检测结果仍呈阳性,而病毒中和试验结果显示该阳性血清稀释度在1512时检测结果就已为阴性,表明该方法的敏感性较高。重复性试验结果显示批内批间变异系数均小于10%,表明该方法重复性较好。采用本实验建立的方法与病毒中和试验同时检测90份牛血清,结果显示二者的符合率为95%。利用本研究建立的间接ELISA方法对188份采自国内两个不同地区的牛血清样品进行了检测,检出BVDV阳性血清162份,阳性血清检出率为86.2%。本研究建立的检测BVDV血清抗体的间接ELISA方法可用于国内BVDV的血清流行病学调查,为相关疫病的防控提供技术支持。 相似文献
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《中国兽医学报》2019,(12):2305-2309
为建立牛环曲病毒快速检测方法,针对牛环曲病毒的保守区域S基因设计合成1对特异性引物,建立了检测牛环曲病毒的RT-PCR方法,并对其进行了敏感性、特异性和重复性检测。结果显示,仅牛环曲病毒阳性模板扩增得到698 bp大小的目的条带,测序结果与GenBank中牛环曲病毒S基因序列(登录号:MG957145.1)的同源性为97%。敏感性试验显示,该方法能检测到的最低核酸含量为1.36×10~4 copies/μL。利用该方法对采集自河南省部分地区的164份临床样品进行检测,结果显示牛环曲病毒的检出率为9.15%(15/164)。结果表明,所建立的牛环曲病毒RT-PCR检测方法灵敏度高、特异性强、重复性好,可用于牛环曲病毒的临床检查和流行病学调查。 相似文献
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试验旨在建立牛副流感3型病毒（bovine parainfluenza type-3 virus,BPIV3）纳米PCR（Nano-PCR）与环介导等温扩增技术（loop-mediated isothermal amplification,LAMP）新型快速分子检测技术,对其进行特异性和敏感性对比试验,并对10份临床样品进行了检测。特异性与敏感性试验结果显示,建立的Nano-PCR和LAMP方法只对BPIV3特异,而对牛呼吸道合胞体病毒、牛传染性鼻气管炎病毒、牛病毒性腹泻病毒无交叉反应;建立的Nano-PCR和LAMP方法具有相同的敏感性,均是普通PCR的10倍,最低核酸检出量均为4.16×102拷贝/μL。临床检测结果显示,建立的两种方法阳性符合率为100%,且阳性检出率均高于普通PCR。因此,本试验建立的Nano-PCR和LAMP方法为BPIV3的临床诊断提供了更快速、敏感、可靠的工具。 相似文献
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根椐GenBank中已发表的牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)、牛冠状病毒(bovine coronavirus,BCV)和牛轮状病毒(bovine rotavirus,BRV)3种病毒基因序列,设计引物。在建立各病毒单项RT-PCR技术的基础上,优化三重RT-PCR反应条件,建立了3种病毒的三重RT-PCR技术,用这3对引物对同一样品中的BVDV、BCV、BRV核酸模板进行三重RT-PCR扩增,结果可同时扩增BVDV的466 bp,BCV的685 bp,BRV的247 bp的特异性片段,而对猪瘟病毒、牛传染性鼻气管炎病毒、牛呼吸道合胞体病毒和牛副流感病毒3型扩增结果均为阴性。敏感性测定表明,该三重RT-PCR技术能检出10 pg的BVDV、1 pg的BRV和10 pg的BCV模板。用45份临床病料对本研究多重RT-PCR技术和单项RT-PCR技术进行对比验证,显示两者的总符合率为100%。表明建立的多重RT-PCR检测方法具有特异、快速、准确的特点,可用于对这3种病毒的同时检测和鉴别诊断。 相似文献
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建立针对牛腺病毒5型六邻体(Hexon)基因的实时荧光定量TaqMan PCR检测方法。根据牛腺病毒5型Hexon基因高度保守区设计引物和TaqMan探针,绘制标准曲线,对其灵敏度、特异性、重复性进行验证,建立牛腺病毒5型实时荧光定量TaqMan PCR快速检测方法。结果显示,构建的重组质粒pUC57-BAV5,标准曲线在10~2～10~7拷贝数之间有较好的线性关系,相关系数为0.975 2,建立检测方法的最低检测限为32拷贝/μL;应用建立的方法检测牛传染性鼻气管炎病毒、牛轮状病毒、牛呼吸道合胞体病毒无交叉反应;重复性试验表明,同一浓度的20个平行样品的变异系数为1.4%。牛腺病毒5型TaqMan探针荧光定量PCR方法具有灵敏度高、特异性强、重复性好等特点,可以用于牛腺病毒5型的快速、准确的检测。 相似文献
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鹅星状病毒RT-PCR检测方法的建立 总被引：1，自引：0，他引：1
《现代畜牧兽医》2021,(4)
为建立一种鉴定鹅星状病毒(GAstV)的RT-PCR方法,试验根据GenBank中GAstV ORF1b基因序列设计1对特异性引物,构建重组质粒pMD18-T-GAstV,以其作为标准品建立GAstV的RT-PCR检测方法。通过对GAstV及其他4种病毒进行检测,优化其反应条件并进行特异性、敏感性和重复性试验以及临床样本检测。结果显示,除GAstV外,鹅细小病毒(GPV)、鸭呼肠孤病毒(DRV)、鸡病毒性关节炎(AVAV)、坦布苏病毒(TmuV)等常见禽病病原利用该方法检测均呈阴性,表明其特异性良好;建立的RT-PCR检测方法的最低检出限可达5.5×10~3,敏感性较高;重复性试验显示其重复性良好。利用该方法对10份人工模拟病料进行检测限LOD分析,阳性样品10倍系列稀释浓度。结果表明,最低检出限可达1.21×10~3copies/μL。研究表明,成功建立了一种鉴定GAstV的RT-PCR方法,且该方法具有快速、廉价、特异性强、敏感性高和重复性好的优点,可用于GAstV的临床诊断。 相似文献
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W L Castleman A Torres-Medina K L Hawkins E J Dubovi J M Atz 《The Cornell veterinarian》1985,75(4):473-483
Severe respiratory disease associated with bovine respiratory syncytial virus (BRSV) infection has been identified in dairy cattle in New York State. The cases identified occurred in dairy calves and heifers. The disease was characterized in 4 animals by pathologic changes including interstitial pneumonia, necrotizing bronchiolitis with multinucleated syncytial epithelial cells and interstitial emphysema. BRSV antigen was demonstrated in lung samples or was isolated in tissue culture in all 4 cases. A retrospective survey of 6279 bovine diagnostic accessions between 1977 and 1982 revealed 66 cases of interstitial pneumonia, often with concurrent bronchiolitis. In this 5 year period, only 1 case in 1981 had interstitial pneumonia and bronchiolitis with pathologic features consistent with BRSV infection. It is concluded that pathogenic BRSV has entered New York State and that it is contributing to clinical respiratory disease in dairy cattle. 相似文献
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Serum samples were collected from early weaned fall calves shortly after the onset of respiratory tract disease. Antibody titers to infectious bovine rhinotracheitis (IBR) virus, parainfluenza type 3 (PI-3) virus, bovine viral diarrhea (BVD) virus, bovine adenovirus type 3 (BAV-3), and bovine respiratory syncytial virus (BRSV) were determined on paired (acute and convalescent) serums. Seroconversion rate (a fourfold or greater rise in antibody titer) for IBR virus was 4.3%, PI-3 virus--16.3%, BVD virus--9.6%, and BAV-3--2.2%. Seroconversion for BRSV was 45.4%. An increased rate of seroconversion for IBR, PI-3, and BVD viruses and BAV-3 was observed in the presence of BRSV seroconversion. These results suggest that BRSV may facilitate infection by other viruses. Results of virus isolation procedures from these calves were negative. 相似文献
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Hazari S Panda HK Kar BC Das BR 《Comparative immunology, microbiology and infectious diseases》2002,25(1):59-68
Seroprevalence of bovine respiratory syncytial virus (BRSV) infection in both exotic and crossbred cattle were described. A baculovirus expressed recombinant purified nucleocapsid (N) protein was used in indirect and sandwich ELISA for screening of 499 bovine sera samples from all over the state for the presence of BRSV antibodies. The seroprevalence rate of BRSV was found to be 46.09% through indirect ELISA while it would found to be 65.33% by sandwich ELISA. The result also indicated that exotic breeds were more susceptible to BRSV infection compared to crossbred cattle. A comprehensive analysis on susceptibility to BRSV as regards to various factors like age and sex was also summarized. 相似文献
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Tegtmeier C Uttenthal A Friis NF Jensen NE Jensen HE 《Zentralblatt für Veterin?rmedizin. Reihe B. Journal of veterinary medicine. Series B》1999,46(10):693-700
During 1 year, the association between microbiological and pathological findings in 72 lungs from calves submitted to the Danish Veterinary Laboratory for diagnostic purposes was studied. All cases were evaluated pathologically and bacteriologically, whereas only 68 cases were examined for the presence of bovine respiratory syncytial virus (BRSV), parainfluenza-3 virus (PI-3 virus) and bovine coronavirus, 62 cases for bovine viral diarrhoea virus (BVD), 45 cases for bovine adenovirus and 51 cases for mycoplasmas. Based on histopathological examination, the cases were diagnosed as fibrinous and/or necrotizing bronchopneumonia, suppurative bronchopneumonia, embolic pneumonia and others. The diagnoses were based on the dominating and most severe lesions in each lung. Haemophilus somnus, Pasteurella multocida, Actinomyces pyogenes, P. haemolytica and BRSV were the most commonly found bacterial and viral lung pathogens, respectively. Pasteurella spp. and H. somnus were often associated with the more severe fibrinonecrotizing type of bronchopneumonia, whereas BRSV was primarily detected in cases of suppurative bronchopneumonia. Mycoplasma bovis was isolated from one case only, whereas M. dispar, M. bovirhinis and Ureaplasma diversum were present, often concomitantly, in the majority of cases. Aspergillus fumigatus was isolated from one case. 相似文献
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Synergistic effects of bovine respiratory syncytial virus and non-cytopathic bovine viral diarrhea virus infection on selected bovine alveolar macrophage functions. 
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下载免费PDF全文 The effect of bovine respiratory syncytial virus (BRSV) and non-cytopathic bovine viral diarrhea virus (ncpBVDV) infection on selected bovine alveolar macrophage (AM) functions was investigated. Alveolar macrophages were harvested from 2- to 6-month-old calves seronegative for BRSV and BVDV and inoculated with approximately 1 median cell culture infective dose of virus per AM. Control, BRSV infected, ncpBVDV-infected and BRSV-ncpBVDV coinfected AM cultures were evaluated for Fc receptor expression, phagosome-lysosome fusion, superoxide anion (O2-) production, and chemotactic activity on Days 1, 3, 5, and 7 post-infection. Both single and combined viral infections significantly depressed AM Fc receptor expression, phagosome-lysosome fusion, and secretion of chemotactic factors with a more significant synergistic depression seen in BRSV-ncpBVDV coinfection. Production of O2- by AM was not decreased by either BRSV or ncpBVDV infection, but was significantly decreased by coinfection with BRSV-ncpBVDV. The present study confirms previous reports of BRSV effects on AM functions and indicate that ncpBVDV affects AM functions in vitro. Coinfection with BRSV-ncpBVDV produced a synergistic depression on AM functions. 相似文献
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A comparison of diagnostic methods for the detection of bovine respiratory syncytial virus in experimental clinical specimens. 总被引：1，自引：0，他引：1 下载免费PDF全文
下载免费PDF全文 K West J Bogdan A Hamel G Nayar P S Morley D M Haines J A Ellis 《Canadian journal of veterinary research》1998,62(4):245-250
Virus shedding was monitored in nasal secretions of 12 calves experimentally infected with bovine respiratory syncytial virus (BRSV) using an antigen capture enzyme-linked immunosorbent assay (ELISA) detecting the nucleoprotein (NP) antigen of BRSV, by a polymerase chain reaction (PCR) amplifying the fusion protein of BRSV, and by a microisolation assay combined with immunoperoxidase staining for the F protein of BRSV. Under the conditions of this study, similar limits of detection and quantitative results were obtained from all three assays. BRSV was detected in nasal secretions of all calves for a minimum of 4 d. Virus shedding began on Day 2 after infection, peaked on Days 3-5, and was cleared in most calves by Day 8. The PCR, and to a lesser extent the ELISA, may detect virus shedding for a longer period after infection than virus isolation, possibly due to neutralization of the virus by rising mucosal antibody. Simulated environmental conditions likely to be experienced during transport of clinical field specimens markedly reduced the sensitivity of virus isolation but had a minimal effect on the results of the NP ELISA. Actual field transport conditions (overnight on ice) had minimal apparent effect on the results of the PCR assay. The less stringent specimen handling requirements, combined with low limits of detection, of both the nucleoprotein ELISA and PCR, indicate either of these assays are more suitable for diagnostic applications than virus isolation. 相似文献
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The detection of bovine respiratory syncytial virus in formalin fixed bovine lung with commercially available monoclonal antibodies and avidin biotin complex immunohistochemistry. 总被引：2，自引：1，他引：1 下载免费PDF全文
下载免费PDF全文 Eight commercially available monoclonal antibodies directed against respiratory syncytial virus antigens were tested for ability to detect bovine respiratory syncytial virus (BRSV) antigen in formalin fixed, paraffin embedded bovine lung using avidin-biotin complex immunohistochemical staining. Monoclonal antibodies from clone 18B2 purchased from Biosoft, Paris, France and those from clone 8G12 purchased from the Department of Veterinary Sciences, University of Nebraska, Lincoln, Nebraska stained BRSV antigen in infected bovine lung with acceptable background staining of uninfected tissues. This method offers advantages over other techniques for BRSV diagnosis in that fresh tissue is not required and all reagents may be purchased commercially. 相似文献
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根椐GenBank中牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)、牛呼吸道合胞体病毒(bovine respiratory syncytial virus,BRSV)和牛副流感病毒3型(bovine parainfluenza virus type 3,BPIV-3)3种病毒基因序列,设计合成引物,建立3种病毒的三重RT-PCR方法。用这3对引物对同一样品中的BVDV、BRSV和BPIV-3核酸模板进行三重RT-PCR扩增,结果显示:可同时扩增BVDV的466 bp,BRSV的735 bp和BPIV-3的258 bp的特异性片段,而对其他4种病原的PCR扩增结果均为阴性;敏感性测定结果表明,该三重RT-PCR技术能检出10 pg的BVDV、1 pg的BPIV-3和10 pg的BRSV模板。用37份临床病料对本研究多重RT-PCR技术和单项RT-PCR技术进行对比验证,结果显示:两者的总符合率为100%。结果表明:建立的多重RT-PCR检测方法,具有特异、快速、准确的特点,可用于对这3种病毒的同时检测和鉴别诊断。 相似文献