共查询到19条相似文献，搜索用时 156 毫秒
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为了对牛呼吸道合胞体病毒(bovine respiratory syncytial virus, BRSV)进行准确定量检测，针对BRSV F基因设计特异性引物，构建pMD19T-BRSV-gF重组质粒，以其为模板优化反应条件及反应体系，建立TB GreenⅡ荧光定量PCR方法并应用于临床BRSV检测。结果显示，针对BRSV F基因建立的TB GreenⅡ荧光定量PCR方法标准曲线线性关系良好，相关系数为0.995 2;灵敏度高，BRSV最低检测限达3.9×101 copies/μL;特异性良好，可特异性检测出BRSV,对牛传染性鼻气管炎病毒和牛病毒性腹泻病毒的扩增呈阴性；重复性好，批内和批间重复性试验变异系数均小于2%。对50份临床样品进行检测，荧光定量PCR对BRSV的阳性检出率(26%)明显优于常规PCR方法(14%)。结果表明针对BRSV F基因建立的TB GreenⅡ荧光定量PCR检测方法适合BRSV的临床检测。 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
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《畜牧与兽医》2019,(12):101-105
为了解吉林省牛病毒性腹泻(BVD)、牛传染性鼻气管炎(IBR)、牛呼吸道合胞体病(BRS)的流行及其病原混合感染情况,在吉林省的9个地区随机采集了325份血清样品,采用ELISA血清抗体检测试剂盒与新型纳米PCR方法检测所采集样品。结果显示,ELISA检测BVDV抗体阳性243份,阳性率为74.77%,IBRV抗体阳性186份,阳性率为57.23%,BRSV抗体阳性90份,阳性率为27.69%,BVDV与IBRV混合感染率为31.69%;BVDV与BRSV混合感染率为9.54%,IBRV与BRSV混合感染率为1.54%,3种病毒混合感染率为17.23%。采用纳米PCR方法检测所有血清显示,BVDV抗原阳性42份,阳性率12.92%,未检出IBRV抗原阳性;BRSV抗原阳性29份,阳性率8.92%。BVDV与BRSV混合感染率为1.85%。 相似文献
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为建立检测牛疱疹病毒（BHV-1）、牛呼吸道合胞体病毒（BRSV）、牛副流感病毒3型（BPIV-3）和牛病毒性腹泻病毒（BVDV）单一或混合感染的荧光PCR检测方法,根据BHV-1 gB基因、BRSV F基因、BPIV-3 M基因和BVDV 5''UTR基因保守区序列分别设计特异性引物和TaqMan荧光探针,经条件优化,成功建立了BHV-1、BRSV、BPIV-3和BVDV的四重荧光PCR检测方法。该方法对牛布鲁氏菌、猪瘟病毒、小反刍兽疫病毒、羊多杀性巴氏杆菌无特异性扩增;对BHV-1、BPIV-3和BVDV的最低检测量均为8.268 copies/μL,对BRSV的最低检测量为82.680 copies/μL;该方法重复性好,CV值为1%~2%。应用本方法检测采自湖南省内某屠宰场的865份样品,结果BHV-1、BRSV、BPIV-3和BVDV等4种病原均有检出,其阳性率分别为0.58%,0.81%,0.23%和0.81%。本研究建立的多重荧光PCR检测方法可同时对BHV-1、BRSV、BPIV-3和BVDV进行检测,为这4种病原的快速诊断和鉴别提供了技术支撑。 相似文献
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为检测商品化猪瘟细胞毒活疫苗中牛病毒性腹泻病毒污染,根据牛病毒性腹泻病毒5′端非编码区基因保守序列设计引物,建立了检测牛病毒性腹泻病毒一步法反转录—聚合酶链(RT-PCR)方法,并对其特异性、敏感性进行了研究。该一步法RT-PCR对牛病毒性腹泻病毒扩增结果为阳性,对照毒株扩增结果均为阴性,对牛病毒性腹泻病毒检测的灵敏性为1pg总RNA量,应用该方法,检测了32批猪瘟细胞毒活疫苗样品,以上结果表明该一步法RT-PCR方法检测速度快、特异性强、敏感性高,可用于猪瘟细胞毒活疫苗中污染的牛病毒性腹泻病毒外源病毒检测。 相似文献
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为建立牛精液中基因I型牛病毒性腹泻病毒(BVDV-1)的快速检测方法,本研究采用Sephycral S-400凝胶对牛精液过滤处理后提取病毒核酸,根据BVDV-1 5'UTR保守区基因序列,设计特异性引物和荧光探针,通过反应条件的优化,建立了牛精液中BVDV-1荧光定量RT-PCR检测方法.该方法可以检测到牛精液中含量为0.0125 TCID50的病毒,灵敏度比病毒分离方法高200倍～2000倍,比常规RT-PCR方法高10倍.对从同一个牛场6个月内采集的120份新鲜牛精液和40份冷冻牛精液用该荧光RT-PCR方法检测,没有检测到BVDV-1阳性样品.对其中的10头牛定期检测精液中病毒的同时,并同步检测了全血中的病毒和血清抗体,结果血清抗体阳性牛精液中没有检测到病毒,本研究结果表明,不能以血清抗体阴阳性做为精液是否带毒的依据. 相似文献
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牛病毒性腹泻病毒一步法RT-PCR检测方法的建立 总被引：1，自引：0，他引：1
根据牛病毒性腹泻/粘膜病病毒5'端非结构蛋白基因序列,设计合成1对特异性引物,建立检测牛病毒性腹泻/粘膜病病毒244 bp片段的RT-PCR一步法。该方法对牛病毒性腹泻/粘膜病病毒NADL、OregonC24V和长春184毒株各标准毒株检测,结果均为阳性。对标准毒株的细胞毒进行检测,其敏感度达0.1 TCID50;而对牛传染性鼻气管炎病毒、猪瘟病毒、牛轮状病毒和牛冠状病毒的细胞培养物进行检测,结果均为阴性。检测460份不同样品,与病毒分离试验比较,符合率100%,证明该方法特异性强,敏感性高。初步结果表明所建立的一步法RT-PCR技术可用于牛病毒性腹泻/粘膜病的检测及流行病学调查。 相似文献
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RT-PCR assays for detection of BRSV, based on four different sets of primers were optimized and evaluated for their sensitivity and specificity. Primers used in this study were specific for genes encoding three BRSV proteins, nucleoprotein N and glycoproteins F and G. Our results indicated that RT-PCR with primers B7:B8 for G protein was the most efficient in detecting BRSV. Starters B7:B8 reacted specifically only with BRSV strains, no cross-reaction with other closely related viruses to BRSV was observed. RT-PCR sensitivity was also high and amounted to 10(1.66) TCID50. Starters for F and N genes of BRSV were not sufficiently specific and cross-reacted with RNA of HRSV. RT-PCR with primers for the genes F and N of BRSV was characterized by a lower sensitivity than RT-PCR with primers B7:B8. In conclusion, RT-PCR specific to a sequence of glycoprotein G gene, seemed to be the most useful for BRSV detection. 相似文献
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Asuka Kumagai Kyoko Kawauchi Kiyohiko Andoh Shinichi Hatama 《Journal of veterinary diagnostic investigation》2021,33(1):162
Bovine respiratory syncytial virus (BRSV) is an etiologic agent of bovine respiratory disease. The rapid evolutionary rate of BRSV contributes to genetic and antigenic heterogeneity of field strains and causes occasional vaccine failure. We conducted molecular epidemiologic characterization of BRSV circulating in Japan to obtain genetic information for vaccine-based disease control. Phylogenetic analysis of G and F gene sequences revealed that all of the isolated Japanese BRSV strains clustered in the same genetic subgroup, which was distinct from the 9 known groups. We assigned the Japanese group to subgenotype X. The Japanese isolates formed 2 temporal clusters: isolates from 2003 to 2005 clustered in lineage A; isolates from 2017 to 2019 formed lineage B. The alignment of the deduced amino acid sequences of the G gene revealed that the central hydrophobic region responsible for viral antigenicity is conserved in all of the isolates; unique amino acid mutations were found mainly in mucin-like regions. Our results suggest that BRSV has evolved uniquely in Japan to form the new subgenotype X; the antigenic homogeneity of the viruses within this group is inferred. 相似文献
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为构建表达牛呼吸道合胞体病毒(BRSV)G蛋白基因的牛疱疹病毒Ⅰ型(BHV-1)重组病毒,本研究将人工合成的BRSV全长G蛋白基因编码序列插入到巨细胞病毒(CMV)启动子之下构建TK基因缺失转移载体。利用磷酸钙-DNA沉淀法将该转移载体与亲本病毒BHV-1/TK-/LacZ+的基因组DNA共转染牛鼻甲细胞后收获增殖的病毒。通过反向蚀斑筛选,得到重组病毒BHV-1/TK-/G+。PCR检测结果证实G蛋白基因已经插入到了亲本病毒BHV-1/TK-/LacZ+的基因组中,间接免疫荧光试验和western blot证实BHV-1/TK-/G+中的G蛋白基因在感染的细胞中获得了表达。本研究为研制BRSV及其他重要牛传染病的BHV-1病毒活载体疫苗奠定了基础。 相似文献
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Obando C Baule C Pedrique C Veracierta C Belák S Merza M Moreno-Lopez J 《Acta veterinaria Scandinavica》1999,40(3):253-262
An investigation based on 2 studies was carried out to assess the involvement of bovine virus diarrhoea virus (BVDV), bovine herpesvirus type 1 (BHV-1), and bovine respiratory syncytial virus (BRSV) in calf respiratory disease in dairy farms in Venezuela. In the first study, 8 farms were selected and paired serum samples from 42 calves with respiratory disease were tested by ELISA for antibodies to the 3 viruses. Seroconversion to BVDV, BHV-1, and BRSV was found to 5, 2, and 6 farms out of the 8, respectively. The proportion of calves that showed seroconversion to BVDV, BHV-1, and BRSV were 19%, 14%, and 26%, respectively. In the second study, another farm having previous serological evidence of BVDV infection was selected. The decline of maternal antibodies against BVDV was monitored in 20 calves and the half-life of maternal antibodies was 34 +/- 12 days presumably indicating an early natural infection with BVDV. Furthermore, sera free of BVDV antibodies that were collected in studies 1 and 2 and were assayed for the presence of BVDV by nested RT-PCR. Two BVDV strains were detected and compared to those of ruminant and porcine pestiviruses. Both strains were assigned to subgroup Ib of type I BVDV. This investigation provides information on BVDV genotypes circulating in Venezuela and may contribute to the establishment of official control programmes against the viruses studied. 相似文献
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BPIV-3和BVDV双重RT-PCR快速检测方法的建立 总被引：1，自引：0，他引：1
参照GenBank中登录的牛副流感病毒3型(BPIV-3)和牛病毒性腹泻病毒(BVDV)全基因序列,分别针对BPIV3特异性NP蛋白保守基因和BVDV保守区段E2基因设计2对引物,经优化反应条件建立了快速鉴别BPIV-3和BVDV的双重RT-PCR诊断方法。最佳扩增条件为94℃30s,56.2℃30s,72℃1min,循环30次;72℃延伸5min,16℃10min;BVDV引物浓度为1.0μmol/L,BPIV-3引物浓度为0.5μmol/L。采用该方法检测BPIV-3和BVDV参考病毒株,能同时扩增出预期为425bp和294bp大小的特异性片段,而扩增牛传染性鼻气管炎病毒、牛合胞体病毒、猪瘟病毒以及牛支原体、致病性大肠埃希菌、多杀性巴氏杆菌A型、化脓隐秘杆菌和鼠伤寒沙门菌等均呈阴性反应。对参考病毒株进行梯度稀释检测,结果证明该方法检测BPIV-3的灵敏度可达10-3 TCID50/0.1mL,而BVDV的灵敏度达102 TCID50/0.1mL。 相似文献
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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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Dung Thi LE Son Vu NGUYEN Mari OKAMOTO Nanako YAMASHITA-KAWANISHI Tung Duy DAO Vuong Nghia BUI Haruko OGAWA Kunitoshi IMAI Takeshi HAGA 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2021,83(8):1273
The detection of bovine foamy virus (BFV) in Vietnamese cattle was performed using conventional PCR targeting pol and gag genes. Out of 243 tested samples, ten (4.1%) and eight (3.3%) samples were positive for BFV gag and pol DNA, respectively. The prevalence of bovine leukemia virus (BLV) estimated by detection of proviral DNA using nested PCR targeting env gene was 26.7% (65/243). The results of nucleotide sequence alignment and the phylogenetic analysis suggested that Vietnamese BFV strains showed high homology to isolates belonging to either European or non-European clades. There was no significant correlation between BLV and BFV. This study provides information regarding BFV infection and confirms the existence of two BFV clades among Vietnamese cattle for the first time. 相似文献