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1.
用digoxigenin标记DNA探针检测鸡贫血病病毒感染   总被引:11,自引:0,他引:11  
用digoxigenin标记鸡贫血病病毒(CAV)衣壳蛋白基因的重组质粒克隆作为探针对江苏、上海等不同地区随机采集的60 ̄120日龄病鸡或死亡鸡的胸腺抽提DNA,进行Dot blot检测CAV感染情况。在被检测的84只鸡胸腺病料中,有24只鸡的病料显示阳性(阳性检出率为28.6%),这一结果表明,CAV感染在我国鸡群中已很普遍,且引起严重的二重感染。  相似文献   

2.
根据鸡传染性贫血病病毒(CAV)的基因结构,设计并合成一对限制扩增长度为0.68kb的PCR引物.直接对临床可疑病鸡的血清进行靶DNA的PCR扩增.在不同地区的3批样品中,有2批扩增出一二分子量接近0.7kb的单一特异性DNA片段.用限制酶BglⅡ消化PCR产物.可获得分子量分别为0.33kb和0.35kb两个DNA片段,其酶切位点与靶DNA的相吻合.将PCR扩增为阳性的病料接种6日龄SPF鸡胚,并取其18日龄胚肝和由接种鸡肛孵出的雏鸡的血清重新进行PCR扩增,结果均为阳性,而同期对照胚肝及雏鸡血清为阴性.此外还用胚胎时接种过病料的雏鸡肝、脾和胸腺等组织抽提液作抗原,建立了检测CAV血清抗体的ELISA方法.研究结果提示,用PCR直接对病鸡血清中CAVDNA的扩增以及CAV抗体的ELISA检测是快速和特异的.它为鸡传染性贫血病(CIA)的流行病学调查、实验室诊断、CAV毒株的分离和鉴定提供了必要的检测手段.  相似文献   

3.
用聚合酶链式反应(PCR)技术,从马立克氏病病毒(MDV)GA株感染的鸡胚成纤维细胞(CEF)基因组DNA中扩增出MDV糖蛋白(gD)抗原基因1209bp编码序列。将该PCR扩增产物于EcoRI和KpnⅠ位点克隆进行pUC18质粒载体中。将gD重组pUC18质粒DNA用Digoxigenin(Dig)标记后,在Southern blot中,该探针能识别MDV基因组DNA的Bam HI-A克隆中的A  相似文献   

4.
凋亡素基因的克隆   总被引:6,自引:1,他引:5  
为了探索应用凋亡素杀死肿瘤细胞的可能性,用PCR方法扩增了鸡贫血病毒(chickenanemiavirus,CAV)的VP3基因(凋亡素)片段,然后将该片段重组于pUC19载体,并进行了限制性内切酶鉴定与序列分析,证实该片段与鸡贫血病毒Cux-1株的VP3DNA序列一致,该DNA全长363bp,编码121个氨基酸。  相似文献   

5.
鸡传染性贫血病(Chicken Infectious Anemia,CIA)是由鸡贫血病毒(Chicken Anemia Virus,CAV)引起的,其特征性病变为骨髓黄化,胸腺萎缩,出现造血机能障碍和免疫机能损害[1,2]。自 1979年从日本发现和分离到 CAV以来[3],德国、英国、美国、澳大利亚、荷兰和以色列都已先后报道了本病的存在。对CAV感染的流行病学调查已表明,世界各地鸡群中,对CAV的感染率都相当高,甚至在曾经认为是SPF鸡群中也有40%-50%表现出CAV抗体阳性。由此可见商品雏鸡中…  相似文献   

6.
产蛋下降综合征(EDS-76)病毒DNA探针的制备及应用研究   总被引:2,自引:0,他引:2  
用纯化的产蛋下降综合征(EDS-76)病毒(H91毒株)悬液提取的核酸,经琼脂糖凝胶电泳只出现1条分子量较大、背景清晰的核酸带。用BamHI酶消化产生4个片段,大小分别为17kb、10kb、4.0kb和2.0kb。将其中的2.0kb片段克隆进pUC18DNA载体中,重组质粒DNA用digoxigenin标记作为DNA探针,在dotblot中该探针不与鹅胚尿囊液核酸抽提物、马立克氏病病毒(MDV)DNA、鸡传染性贫血病毒(CAV)DNA、SPF鸡基因组DNA等核酸反应,只与3株EDS病毒(EDS标准毒株AV-127、EDSH91毒株和从健康鹅体中分离的EDSY81毒株)DNA呈阳性反应。人工感染产蛋母鸡和雏鸡后24h即可用该探针从泄殖腔棉拭子样品中检测出EDS病毒DNA,在感染后35h仍能从部分感染鸡样品中检出。对部分探针检测阳性鸡的泄殖腔棉拭子样品用SPF鸡胚分离病毒,并用HA和HI试验检测分离病毒的特异性;在感染过程中,同时检测了血清中HI抗体的消长情况。结果表明,人工感染鸡排毒至少可以维持2个月左右,而且血清中HI抗体即使很高,也不能阻止感染鸡的排毒。  相似文献   

7.
IBDV VP2/VP243基因DNA疫苗表达质粒的构建与表达   总被引:2,自引:0,他引:2  
将传染性法氏囊病病毒(IBDV)VP2/VP243插入真核表达载体pcDNA中,置于CMV启动子的调控之下,构建成DNA疫苗表达质粒pcDNA VP2和pcDNA VP0。分别转染CEF细胞后,于24、48、72h取细胞裂解液进行ELISA、SDS-PAGE和Western blot检测,pcDNA VP2于转染后24h呈阳性反应,48h表达量高于24h;pcDNA VP0于转染后48h呈阳性反应  相似文献   

8.
鸡贫血病毒DNA的PCR扩增及其分子克隆   总被引:4,自引:0,他引:4  
用PCR方法扩增了国内分离的鸡贫血病毒(CAV)SJ1株的含VP2、VP3以及部分VP1的1500bpDNA片段。经限制性内切酶酶切分析,该片段与Cux-1株的酶谱相似。同时,以pUC119质粒载体克隆了这一DNA片段。  相似文献   

9.
北京地区鸡群网状内皮组织增殖病感染的血清学调查研究   总被引:12,自引:1,他引:11  
本文通过制备纯化的禽网状内皮组织增殖病病毒(REV)和SPF鸡抗REV的特异血清,成功地建立了间接酶联免疫吸附试验(ELISA),首次对北京地区种鸡群REV的感染情况进行了血清学调查。对有疑似临床症状的鸡作重点采样,从7个鸡场112份血清样品中检出54只阳性鸡,阳性率为48.2%。从11个鸡场随机抽样204份,检出阳性鸡28只,阳性率为13.7%。结果发现,雏鸡、中鸡的感染率较成鸡低,祖代鸡的感染率较父母代低。感染情况似乎与鸡的品种无关。大多数感染鸡群显示不出该病的临床症状或明显的生产障碍。试验结果表明,间接ELISA方法检测REV血清抗体,具有简便易行、敏感性高、特异性强、重复性好等优点,可作为检测REV血清抗体的比较理想的方法  相似文献   

10.
为了建立一种监测鸡群马立克氏病病毒(MDV)早期感染的微量PCR方法,根据血清Ⅰ型马立克氏病病毒(MDV1)基因组BamHI-L片段的核酸序列设计了1对寡核苷酸引物,用含0.3UTaq酶的10μL反应体系分别扩增了MDV1型(京-1株、MD11/75c株)、MDV2型(SB-1株)和HVT(FC-126株)的核酸,并用京-1株感染细胞DNA作了敏感性试验。结果显示,该引物对MDV1型病毒特异,京-1株和MD11/75c株都扩增到425bp的核酸条带,而SB-1株、FC-126株和正常CEF细胞的DNA都没有得到任何条带;敏感性试验结果表明,微量PCR能够检测到0.1pg的京-1株感染细胞DNA;对京-1株感染鸡,在接种后第5天就能在血液细胞中检测到MDVDNA。微量PCR可望成为监测鸡群MDV早期感染的一种快速、有效的方法。  相似文献   

11.
应用能特异性扩增出鸡传染性贫血病毒( C A V) 058 kb D N A 的已知引物,对江苏某地区疑为 C A V感染的 15~30 日龄病鸡的肝 D N A 样品进行了 P C R 扩增。结果,在被检的 20 份样品中,有 6 份为 P C R 阳性,阳性率 30% 。利用地高辛标记的 085 k b 的 C A V 核酸探针对相同样品进行斑点杂交,结果与 P C R 扩增相同。对应的病鸡血清经间接免疫荧光试验( I I F A)发现,有 7 份为 C A V 抗体阳性,与 P C R 扩增结果的符合率为 77% 。初步结果表明,江苏某地区存在 C A V 感染, I I F A 与 P C R 的结果有差异  相似文献   

12.
The pathogenesis of chicken infectious anaemia virus (CAV) infection was studied in 6-week-old and one-day-old SPF chickens inoculated intramuscularly with graded doses of Cux-1 strain (10(6)-10(2) TCID50/chicken). Viraemia, virus shedding, development of virus neutralizing (VN) antibodies and CAV distribution in the thymus were studied by virus isolation, polymerase chain reaction (PCR), immunocytochemistry (IP) and in situ hybridization until postinfection day (PID) 28. In 6-week-old chickens infected with high doses of CAV, viraemia and VN antibodies could be detected 4 PID and onward without virus shedding or contact transmission to sentinel birds. However, virus shedding and contact transmission were demonstrated in one-day-old infected chickens. In the 6-week-old groups infected with lower doses, VN antibodies developed by PID 14, transient viraemia and virus shedding were detected. The thymus cortex of all 1-day-old inoculated chickens stained with VP3-specific mAb. Cells with positive in situ hybridization signal were fewer and scattered throughout the thymus tissue of the one-day-old inoculated chickens as compared to IP-positive cells. These results suggest that early immune response induced by high doses of CAV in 6-week-old chickens curtails viral replication and prevents virus shedding.  相似文献   

13.
Direct detection of chicken anemia virus (CAV) DNA in tissues and sera was investigated by a polymerase chain reaction (PCR) assay. Using a pair of primers constructed to amplify the coding sequence of the CAV DNA genome, the PCR assay was shown to be extremely sensitive, being able to detect 1 fg of CAV replicative form DNA. The oligonucleotide primers used for the PCR yielded 583 base-pair (bp) amplified product, which was sized by ethidium bromide-agarose gel electrophoresis. Tissue samples from seven cases of suspected chicken infectious anemia were obtained for CAV isolation. DNA extracted from the homogenized suspension of pooled tissues of each case was analyzed by the PCR assay. Results showed that five of the seven cases were positive for CAV DNA by PCR, whereas CAV was isolated from four cases only. The PCR assay also detected CAV DNA in two of 37 serum samples from disease-free chickens. The specificity of PCR was confirmed by chemiluminescence dot-blot analysis of the amplified products.  相似文献   

14.
为评价PCR结合斑点杂交技术在鸡REV检测中的应用价值,采用PCR法制备禽网状内皮细胞增生症病毒特异性地高辛标记DNA探针,同时用PCR技术、斑点杂交方法和PCR产物斑点杂交方法检测了不同地区的病、死鸡的组织样品REV的感染情况。结果表明,PCR产物斑点杂交法的检出率(45.16%,14/31)高于组织DNA直接斑点杂交法(32.26%,10/31),显著高于单纯PCR扩增法(0%,0/31)。PCR结合斑点杂交检测技术能快速、敏感、准确,充分避免PCR中的假阴性和假阳性现象,值得推广应用。  相似文献   

15.
鸡贫血病毒VP1和VP2蛋白在家蚕中的联合表达   总被引:1,自引:0,他引:1  
将鸡贫血病毒VP1和VP2基因分别克隆入转换载体pBacPAK8中,获得重组转移质粒pBac-vp1和pBac-vp2。以上两质粒分别与CvnⅠ酶切线性化的亲本病毒Bm-BacPAK6DNA共转染家蚕细胞,通过蓝白斑筛选,纯化得到重组病毒Bm-vp1和Bm-vp2。PCR分析表明Vp1和Vp2基因已整合进杆状病毒基因组中。将Bm-vp1和Bm-vp2共感染5龄家蚕,通过表达产物免疫SPF鸡产生的抗血清与CAV感染的MDCC-MSB1细胞的间接荧光抗体分析,证明表达产物能诱导鸡产生相应的抗体。该研究表明,表达VP1和VP2蛋白的重组家蚕杆状病毒(recombinant BmNPV)是很有前途的CAV亚单位疫苗的生产系统。  相似文献   

16.
山东省鸡传染性贫血流行病学调查   总被引:1,自引:0,他引:1  
本文对山东省12个地市58个鸡群传染性贫血的流行情况进行了调查,共检测了368份血清样品,蛋鸡群、肉鸡群和种鸡群的鸡传染性贫血病毒(CAV)ELISA抗体检出率分别为91.7%、77.6%和84.78%,平均抗体阳性率为83.2%,结果表明近几年山东省鸡群中CAV感染相当普遍,而且蛋鸡和种鸡抗体阳性率明显高于肉鸡群。  相似文献   

17.
A fluorescence quantitative PCR assay was developed using primers and probes designed according to a high conservative sequence of chicken infectious anemia virus (CAV). Detection of viral DNA copy number was determined by extrapolation from a CAV plasmid-based standard curve. The results showed that the estimated average viral copy number could detect 1.8×101 copies/μL of plasmid DNA with high reproducibility (CAV of the intra or inter) assay was below 2%. Furthermore, 1 day-old SPF chicken were inoculated with CAV and the virus loads were detected in multiple organs. The results showed that the virus could be detected in the brain, heart, liver, spleen, lung, kidney, thymus, bursa and serum and the virus loads in these organs showed no significant difference. No cross-reaction was detected with other avian virus.  相似文献   

18.
分别用digoxigenin标记的鸡传染性贫血病毒(CAV)和禽网状内皮增生症病毒(REV)核酸探针以及PCR技术对某种鸡场进行检测,检测结果表明,在20个样品中,CAV和REV感染的阳性率分别为10%和5%,这一结果显示,CAV和REV感染是导致该鸡场发生免疫抑制的主要病因之一。  相似文献   

19.
Chicken anemia virus (CAV) was isolated for the first time from the Nigerian chicken population. The virus was recovered from necropsied birds from broiler and pullet flocks that suffered disease outbreaks tentatively diagnosed as infectious bursal disease. A sensitive polymerase chain reaction (PCR) assay detected CAV DNA in tissues of necropsied birds. Restriction endonuclease analysis performed with the 733-bp PCR product and the Cfo I enzyme indicated at least two different CAVs were circulating among the Nigerian chicken population. Four isolates were obtained from pooled liver and thymus tissues using the MDCC-MSB1 cell line. These isolates were found to be antigenically closely related to the Cuxhaven-1 (Cux-1) reference strain of CAV when reacted with four monoclonal antibodies prepared against the Cux-1 virus. One of the isolates (isolate A) induced thymus atrophy, bone marrow aplasia, and low hematocrit values when inoculated into 1-day-old specific-pathogen-free chickens. These findings not only demonstrate that CAV is present in Nigeria, but they also likely represent the first cell culture isolation of the virus in Africa.  相似文献   

20.
鸡贫血病毒实验感染雏鸡胸腺与骨髓的超微结构观察   总被引:2,自引:1,他引:1  
用鸡贫血病毒 Cux 1 毒株接种于 1 日龄 S P F 雏鸡,在确认感染成功的基础上,对感染雏鸡胸腺和骨髓的超微结构进行了观察。电镜观察表明,感染鸡骨髓和胸腺细胞的形态变化具有较典型的凋亡特征,如细胞膜出泡、核染色质凝结边集、细胞核裂解、出现凋亡小体等,此外还见有可能由病毒粒子形成的环状物。本试验为证明鸡贫血病毒能引起细胞凋亡提供了依据。  相似文献   

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