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1.
鸡致病性外源性禽白血病病毒特异性核酸探针交叉斑点杂交检测试剂盒的研制 总被引：1，自引：0，他引：1
用特定引物通过PCR合成了经地高辛标记的鸡致病性外源性及内源性禽白血病病毒特异性核酸探针,通过交叉斑点分子杂交,这些探针将可用于检测病料样品中致病性外源性禽白血病毒特异性核酸的存在。利用此试剂盒,对从病料组织样品中提取的基因组DNA作交叉斑点分子杂交或对提取的DNA用相应引物扩增后的PCR产物作交叉斑点分子杂交,可在24～36h内完成检测并报告结果。 相似文献
2.
本试验根据GenBank中登录的禽白血病病毒(ALV)基因组序列,设计合成了2对引物,外部引物的扩增片段大小为478 bp,内部引物的扩增片段大小为314 bp,建立了适合ALV快速检测的套式PCR方法(nested-PCR)。采用该方法对ALV毒株进行了检测,试验结果表明,能扩增到314 bp的条带,禽流感病毒(H9亚型)、新城疫病毒、传染性法氏囊病病毒、减蛋综合征病毒、禽网状内皮增生病病毒、禽呼肠孤病毒、马立克氏病病毒的扩增结果均为阴性。该方法第1次扩增的敏感性是100 pg,第2次扩增的敏感性是1 fg,第2次比第1次扩增的敏感性高105倍。所建立的套式PCR方法具有敏感性高、重复性好、特异性强等优点,可用于禽白血病病毒(ALV)的临床诊断和分子流行病学调查等。 相似文献
3.
我国部分地区蛋鸡群ALV-J及与REV、MDV、CAV混合感染检测 总被引：7，自引：1，他引：6
为了解J亚群禽白血病病毒(ALV-J)及其与禽网状内皮细胞增生症病毒(REV)、马立克氏病病毒(MDV)和鸡传染性贫血病病毒(CAV)的混合感染现象,本研究从宁夏、湖北、广东、山东、辽宁、吉林、黑龙江7个省的39个蛋鸡群收集临床表现和剖检病理变化疑似禽白血病的病料样品184份,采用PCR、病毒分离和IFA检测样品中ALV-J、REV、MDV和CAV。结果表明,7个省蛋鸡场均存在ALV-J感染,病料样品阳性率为60.9%,检测鸡群阳性率为82.1%,与REV、MDV、CAV的混合感染率分别为13.6%、24.5%、22.8%,其中存在较为严重的双重感染(29.0%)和3重感染(18.8%),甚至4重感染(1.7%)。研究结果表明,我国蛋鸡群中普遍存在ALV-J感染,而且与REV、MDV、CAV混合感染严重;提示ALV-J已经可以引起蛋鸡群发病,在临床诊断和致病性研究中,应考虑到多重感染的影响。 相似文献
4.
Detection and localization of naturally transmitted avian leukosis subgroup J virus in egg-type chickens by in situ PCR hybridization 总被引：1，自引：0，他引：1
Li N Xu B Dong W Qiao S Lee LF Zhang HM Li M Du N 《Journal of veterinary medicine. A, Physiology, pathology, clinical medicine》2007,54(10):553-558
Avian leukosis virus (ALV) subgroup J (ALV-J) is an exogenous ALV and causes myeloid leukosis in meat-type chickens. We have previously reported the isolation and identification of ALV-J in commercial layer flocks from 12 farms in northern China. In this report, we further characterized this virus by in situ polymerase chain reaction (PCR) hybridization in various affected organs of chickens from six of the 12 farms. A routine method for hybridization of nucleic acid uses radioactive probe, such as a P32-labelled probe. We found that the non-radioactive digoxigenin (DIG) probe is sensitive enough to detect the nucleic acid of virus in chicken tissues. We used a pair of published primers (H5/H7) specific to the gp85 envelope gene and 3' region of pol gene of prototype ALV-J strain HPRS-103. The total RNA extracted from tumour, bone marrow, oviduct, liver and spleen of the diseased chickens from six commercial flocks, and cDNA was successfully amplified. Using the primers and cDNA, we obtained an ALV-J-specific cDNA probe of 545 bp in length by PCR. In situ PCR with H5/H7 primers was carried out in the paraffin sections from tissues of the diseased chickens, followed by in situ hybridization using the DIG-labelled cDNA probe. Positive hybridization signals were detected in the cytoplasm of paraffin sections of tumours and other organ tissues. The intensity of the signals was documented using an image analysis system measuring integral optical density (IOD). The IOD values for tissue sections treated by in situ PCR hybridization are significantly higher than that by in situ hybridization alone (P < 0.01). These data taken together suggest that in situ PCR hybridization is a more sensitive technique for detection of ALV-J in tissue sections. 相似文献
5.
Feather pulp from experimentally infected chickens was used as a source of DNA for polymerase chain reaction (PCR) amplification of avian leukosis virus subgroup J (ALV-J) proviral DNA. A primer set that produces a large amplicon (approximately 2,125) was used to detect ALV-J proviral DNA. This primer set was used in lieu of previously published primers because it allows for sequencing of the entire envelope gene and because it was able to detect diagnostically a number of North American ALV-J isolates that could not be detected with previously published primers and PCR conditions. ALV-J proviral DNA was detected in feather pulp at 7 days of age in more than 90% of birds infected as embryos and 7 days postinoculation in over 50% of chickens infected at 3 days of age. The results obtained with PCR on feather pulp were compared with those of virus isolation. In the embryo-inoculated birds, the percentages of agreement between PCR and virus isolation were 92.5% at 7 days of age and 100% at 28, 42, 49, and 56 days of age. However, the overall sensitivity of virus isolation in embryo-infected birds was higher, particularly at 7 and 56 days of age. In chickens inoculated at 3 days of age, the percentages of agreement of detection between PCR and virus isolation ranged from 75% at 10 days of age to 100% at 42 days of age. Agreement of negative results of ALV-J detection by PCR and virus isolation in chickens infected posthatch ranged between 66.6% and 100% between the ages of 10 and 42 days. Virus isolation requires chicken embryo fibroblasts of specific genetic lines, and the process takes onaverage 7-9 days. Aseptic collection of blood and tissues for virus isolation and molecular detection of ALV-J requires sterile necropsy instruments as well as syringes and needles for each individual chicken, whereas sterile microcentrifuge tubes and gloves are the only equipment necessary for aseptic feather pulp collection for ALV-J detection by PCR. PCR-based detection of ALV-J in feather pulp is especially suitable when ALV-J infection must be diagnosed rapidly and unequivocally without killing the chicken(s) and in situations where crucial reagents or suitable virus propagation substrates are not readily available for isolation and propagation of ALV-J in cell culture. 相似文献
6.
Sironi G Manarolla G Pisoni G Recordati C Rampin T 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2006,53(7):347-349
The study describes a highly productive myotropic avian leukosis virus infection (ALV) in a 3-month-old female chicken. At necropsy, ascites, hepatic fibrosis and cardiomegaly were seen. Histologically, the most striking lesion was the presence of cytoplasmic basophilic inclusions in myocardial fibers. Immunostaining for ALV group specific antigen p27 revealed a diffuse presence of virus antigen in cardiac myofibers, in smooth muscle fibers of most of the organs, and in rare, pancreatic and ovarian theca cells. Ultrastructurally, myocardial inclusions consisted of clusters of 50-60 nm round particles with interspersed ribosome-like granules. Numerous C-type particles were found in intercellular spaces of ALV p27 positive tissues. PCR analyses revealed the presence of both ALV-E and ALV-J related sequences. In chicken genome, ALV-E is usually present as endogenous provirus therefore, the pathological findings observed in this case are considered to be related with the ALV-J infection. The results of this report further confirm that ALV-J may be responsible for highly productive myotropic infections. 相似文献
7.
为了解广东地方品种鸡J亚群禽白血病病毒(ALV-J)净化情况,利用ALV-p27特异性抗原检测、病毒分离、PCR扩增和全基因组测序分析等方法,从广东某地方品种鸡群中分离鉴定出1株ALV-J,命名为GDYH-Y1。全基因组序列分析表明,GDYH-Y1分离株的LTR、gag和pol基因相对保守,而3′UTR和gp85基因变异较大,其中gp85基因与ALV-J参考毒株序列同源性仅为87.5%~92.8%,3′UTR区的rTM出现大量碱基缺失,遗传进化分析表明GDYH-Y1分离株与美国白羽肉鸡源分离株ADOL-7501亲缘关系最近。研究为广东地方品种鸡ALV-J净化效果提供数据参考,为分析广东地方品种鸡ALV-J毒株的分子特征和变异趋势提供了重要的数据资料。 相似文献
8.
为挖掘感染ALV-J汶上芦花鸡的肝差异表达致病相关基因。本研究以汶上芦花鸡为试验素材,分别在42和300日龄进行ALV血液病毒分离检测筛选ALV阴性和阳性个体,对ALV阳性个体有病理变化的肝组织和阴性个体肝组织进行PCR检测,分别选取3只ALV阳性（G1组）和阴性（G2组）个体的肝组织,并对G1组PCR产物测序、聚类分析确定ALV亚型,利用RNA-Seq测序技术筛选差异表达基因,并进行功能分析,利用荧光定量qRT-PCR对部分差异表达基因进行验证。结果表明,G1组样品经PCR检测、PCR扩增产物测序和聚类分析确定ALV为J亚型,转录组分析发现,共有42个差异基因在GO和KEGG中富集,其中,上调基因18个,下调基因24个。随机选取的5个差异表达基因qRT-PCR验证结果与RNA-Seq测序结果相一致。GO分析显示,差异表达基因主要涉及细胞过程、代谢过程、对刺激的反应、免疫系统等;KEGG分析显示,差异表达基因主要富集在细胞过程、信号传导、疾病、新陈代谢等信号通路。本研究通过转录组分析发现了影响ALV-J致病性的多个基因和关键信号通路,为深入了解ALV-J致病的分子机制提供了思路。 相似文献
9.
High virus titer in feather pulp of chickens infected with subgroup J avian leukosis virus 总被引：1，自引：0，他引：1
Subgroup J avian leukosis viruses (ALVs), which are a recombinant virus between exogenous and endogenous ALVs, can spread by either vertical or horizontal transmission. Exogenous and endogenous ALVs can be detected in feather pulp. In this study, virus titers in feather pulp of chickens infected with subgroup J ALV were compared with those of plasma and cloacal swab. All of the broiler chickens inoculated with subgroup J ALV at 1 day old were positive for virus from feather pulp during the experimental period of between 2 wk and 8 wk of age. Virus titers in feather pulp of some broiler chickens infected with subgroup J ALV were very high, ranging from 10(7) to 10(8) infective units per 0.2 ml. Virus titers in feather pulp were usually the highest among the samples of plasma, cloacal swab, and feather pulp tested. In another experiment in which layer chickens were inoculated with subgroup J ALV at 1 day old, virus was detected in feather pulp from 2 wk until 18 wk of age, and virus persisted longer in feather pulp than in plasma. Almost all of the layer chickens tested were positive for virus by polymerase chain reaction (PCR) with DNA extracted from feather pulp samples at 2, 4, and 10 wk of age, and the PCR from feather pulp was more sensitive than virus isolation from plasma, cloacal swab, and feather pulp. All above results indicate that samples of feather pulp can be useful for virus isolation and PCR to confirm subgroup J ALV infection. 相似文献
10.
随机抽取安徽省3个肉种鸡场种鸡及3个地市农贸市场商品鸡的血清样本和泄殖腔拭子,采用ELISA进行鸡白血病检测,并对J亚群鸡白血病病毒抗体阳性鸡群中病鸡的肝组织、全血及上毒细胞提取DNA进行PCR扩增和基因测序。检测结果表明,3个肉种鸡场中J亚群鸡白血病抗体阳性检出率分别为：0、73.50%和15.22%;3个农贸市场商品鸡抗体阳性检出率分别为：12.00%、2.22%和9.10%。3个肉种鸡场A、B亚群鸡白血病的抗体阳性率分别为0、17.93%和1.08%,ALV抗原阳性率分别为2.17%、17.93%和15.22%。PCR检测结果表明,从可疑发病鸡的肝组织和全血中均能扩增出ALV-J gp85特异性片段。结果证实安徽省鸡群中有ALV-J感染,并呈不同程度的流行,应引起高度重视。 相似文献
11.
2009年我国部分地区禽白血病分子流行病学调查 总被引：12，自引：3，他引：9
为了解自2009年年初以来国内一些地区禽白血病流行情况及流行毒株的分子特征,我们从湖北、黑龙江、山东、辽宁、吉林、广东、宁夏、安徽8个省区39个鸡场采集疑似禽白血病病料样品178份,用ALV-A、ALV-B和ALV-J特异性引物,通过PCR方法进行检测。结果表明,8个省的35个鸡场的124份病料中检出了ALV-J(69.7%);25份病料中检出了ALV-A(13.9%);7份病料中检出了ALV-B(3.9%)。14个分离毒株env基因氨基酸同源性为84.3%～99%;与J亚群原型毒株HPRS-103的氨基酸序列同源性为87.3%～98.2%;与其它J亚群env基因氨基酸序列同源性为83%～97.4%。遗传进化分析表明,14个ALV-J分离株分别分属于不同的分支。其中,LJL09DH02分离株与其它分离株及参考毒株的的亲缘关系最远,与HPRS-103的氨基酸同源性仅为87.3%。另外4个分离株的env基因与HPRS-103的氨基酸同源性低于93%,其余9株与HPRS-103的同源性较高(96.6%以上)。该调查结果表明,我国目前ALV的感染主要以J亚群为主,ALV-A和B同时存在。 相似文献
12.
OBJECTIVE: To determine the extent of avian leukosis virus subgroup J (ALV-J) infection in Australian broiler breeder flocks, using virus isolation and molecular biological detection. Any resultant ALV-J viral isolates to be characterised by neutralisation cross testing in order to determine antigenic relationships to overseas isolates of ALV-J. STUDY DESIGN: Samples of blood, feather pulp, albumen and tumours were obtained from broiler breeder flocks which represented four genetic strains of meat chickens being grown in Victoria, South Australia, NSW and Queensland. Dead and ailing birds were necropsied on farm and samples were collected for microscopic and virological examinations. Virus isolation was carried out in C/O and DF-1 CEF cultures and ALV group specific antigen was detected in culture lysates using AC-ELISA. Micro-neutralisation assay was used for antigenic characterisation of selected isolates. Genomic DNA was isolated from cultured cells, tumours and feather pulp. ALV-J envelope sequences were amplified by PCR using specific ALV-J primers while antibodies against ALV-J were detected by ELISA. RESULTS: A total of 62 ALV-J isolates were recovered and confirmed by PCR from 15 (31.3%) of 48 breeder flocks tested. Antibody to ALV-J was detected in 20 (47.6%) of the 42 flocks tested. Characteristic lesions of myeloid leukosis caused by ALV-J were found in affected flocks. The gross pathological lesions were characterised by skeletal myelocytomas located on the inner sternum and ribs, neoplastic enlargement of the liver, and in some cases gross tumour involvement of the spleen, kidney, trachea, skeletal muscles, bone marrow, skin and gonads. Microscopically, the tumours consisted of immature granulated myelocytes, and were present as focal or diffuse infiltrations in the affected organs. Virus micro-neutralisation assays demonstrated antigenic variation among Australian isolates and to overseas strains of ALV-J. CONCLUSION: ALV-J infection was prevalent in Australian broiler breeder flocks during 2001 to 2003. Australian isolates of ALV-J show a degree of antigenic variation when compared to overseas isolates. 相似文献
13.
本试验根据GenBank中登录的禽传染性贫血病毒(CAV)基因序列,设计合成2对引物,外引物的扩增片段大小为485 bp,内引物的扩增片段大小为297 bp,建立了适合CAV快速检测的套式PCR方法(nested PCR),并采用该方法对CAV阳性毒株及临床病料进行了检测。结果显示,该方法能扩增到297 bp的条带,禽流感病毒(H9亚型)、新城疫病毒、传染性法氏囊病病毒、禽网状内皮增生病病毒、减蛋综合征病毒、禽呼肠孤病毒、马立克氏病病毒的扩增结果均为阴性。该方法第1步扩增的敏感性是100 pg,第2步PCR扩增的敏感性是1 fg,敏感性提高了105倍。本研究建立的CAV套式PCR方法具有敏感性高、重复性好、特异性强等优点,可用于CAV的临床诊断和分子流行病学调查等。 相似文献
14.
为研究HR土鸡中存在的不同亚型禽白血病病毒(ALV)共感染的情况,本实验分别采集46只HR土公鸡的泄殖腔棉拭子和455枚鸡蛋卵白样品,采用ELISA试剂盒检测p27抗原;并采用相应的ELISA试剂盒分别检测卵黄中J亚型ALV(ALV-J)和AB亚型ALV(ALV-AB)抗体。结果表明:HR土公鸡泄殖腔棉拭子样品中p27检出阳性率为87%(40/46),卵白检出率为74.7%(340/455);而卵黄中ALV-J和ALV-AB抗体阳性率分别为0(0/30)和80%(24/30)。无菌采集初步筛选p27抗原检测为阳性的5只HR土公鸡的抗凝血接种CEF,采用抗ALV-J和ALV-A的单克隆抗体进行IFA检测,结果显示5份样品中ALV-A和ALV-J的阳性率均为100%(5/5)。同时选取HR土鸡分离株HR332进行PCR扩增鉴定,结果表明分离株HR332存在ALV-J(HR332J)和ALV-A(HR332A)。其中,HR332J与11株ALV-J国内外参考株的同源性为92.4%～97.9%;HR332A与ALV-A参考株RSA-A、MQNCSU的同源性分别为90.1%和89.7%,与国内分离株SDAU09E2的同源性为99.0%。本研究显示,地方品种HR土鸡存在不同亚型ALV共感染,同时ALV-A和ALV-J共感染同一个鸡的现象已经存在。 相似文献
15.
为建立一种H3亚型禽流感病毒(AIV)的检测方法,本研究针对H3亚型AIV HA基因保守序列,设计并筛选出2对特异性引物,通过优化反应条件,建立了H3亚型AIV巢式PCR检测方法。对该法进行特异性和敏感性检验,并用该法对96份临床样品进行检测。特异性试验结果表明该法只能检测到H3亚型AIV,对其他常见禽病病原体不扩增;敏感性试验结果表明该巢式PCR对H3亚型AIV检测下限为1×103拷贝/μL,灵敏度比常规PCR高100倍;96份临床样品检测结果与病毒分离结果一致。本研究所建立的巢式PCR为H3亚型AIV的诊断提供了一种准确、有效的检测方法。 相似文献
16.
为了解禽白血病病毒（ALV）贵州流行株的遗传变异情况及分子特征,本试验基于ALV env基因设计合成引物对禽白血病贵州临床病例进行目的基因扩增、克隆和序列分析。结果显示,从临床病例中筛选获得3份阳性样本,PCR扩增均获得大小约921 bp的目的基因片段,将其命名为：GZ-ALV-1株、GZ-ALV-2株和GZ-ALV-3株。序列分析结果显示,3株ALV贵州流行株之间核苷酸同源性在97.2%~97.6%之间,与国内外ALV-J的同源性相对较高,为93.1%~99.3%;而与A、B、C、D、E、K亚群ALV同源性仅为51.4%~53.2%。系统进化分析显示,3株ALV贵州流行株与ALV-J亚群参考株处于同一分支,表明本试验所检测的ALV毒株均为ALV-J亚群;与A、B、C、D、E、K亚群处于不同进化分支。基因变异分析显示,3株流行株37处相同核苷酸变异导致17处氨基酸发生位点变异,其中9个可变点在高变区hr1和hr2,1个可变点在低变区vr3。结果表明,3株ALV贵州流行株均为ALV-J亚群,env基因存在位点发生了变异,且可变位点位于序列高变区。本研究结果为明确贵州禽白血病流行概况及ALV的防控与净化提供基础数据。 相似文献
17.
2009年8月,山东省邹城市某海兰褐蛋鸡群,160日龄发病,死亡率为7％.患鸡经大体剖检、病理组织学、PCR和免疫组织化学等检测,确诊为禽白血病病毒J亚群(ALV-J)感染.病理组织学检测发现,病鸡单独患血管瘤,或髓细胞瘤和纤维肉瘤多发性出现,由ALV-J自然感染引起同一鸡体出现髓细胞瘤和纤维肉瘤尚属国内外首次报道.肝脏研磨接种DF-1细胞培养7d后传3代,细胞无病变,ELISA检测感染细胞上清ALV p27抗原阳性,进一步确诊此鸡群为ALV感染.对病变严重的鸡进行病毒分离及ALV-J gp85基因同源性比较显示与原型株HPRS-103的同源性最高,达94.1％.本研究丰富了ALV-J感染的临床诊断依据,并为ALV-J在我国蛋鸡群中多潜能致瘤机制的研究提供了科学基础. 相似文献
18.
Development and characterization of monoclonal antibodies to subgroup J avian leukosis virus. 总被引：6，自引：0，他引：6
In an attempt to develop a specific diagnostic test for avian leukosis virus (ALV) subgroup J (ALV-J) strain Hc1, four monoclonal antibodies (MAbs), JE9, G2, 145, and J47, were generated that are specific for ALV-J envelope glycoprotein, gp85. Polymerase chain reaction (PCR) was used to amplify genomic pro-viral DNA of Avian Disease and Oncology Laboratory (ADOL)-Hc1 and ADOL-4817 envelope genes. Both open reading frames encoding glycoproteins gp85 and gp37 were cloned into baculoviruses. Abundant expression of gp85 and gp37 was detected in the recombinant viruses with specific antibody to Hc1 strain of the ALV-J. The expressed proteins were used for immunization of mice to produce hybridoma cell lines secreting MAbs specific to ALV-J envelope protein. A panel of MAbs was generated by fusing NS1 myeloma cells and spleen cells from mice immunized with the recombinant baculoviruses. With the use of an immunofluorescence assay, three MAbs (JE9, G2, 145) reacted with ALV-J but not with subgroups A, B, C, D, or E of ALV. MAb J47 reacted with all exogenous subgroups of ALV including A, B, C, D, and J but not with endogenous subgroup E viruses. Western blot analysis was performed with all four MAbs against recombinant baculovirus and Hc1-infected chicken embryo fibroblast (CEF) lysates. A major band with a molecular weight about 90 kD corresponding to the size of ALV-J envelope was consistently obtained. With these MAbs, we detected the Hc1 antigen in CEFs infected with several ALV-J viruses isolated in the United States and also in tissue sections from chickens infected with Hc1 strain of ALV-J. These MAbs will be useful reagents for the diagnosis of ALV-J infection because they recognize a common antigenic epitope in six isolates tested thus far. 相似文献
19.
血管瘤相关J亚群禽白血病病毒ZH-08株的分离与全基因组序列测定 总被引：3，自引：0，他引：3
本研究从临床表现为典型血管瘤型禽白血病病例的广东某肉种鸡场的病鸡中,分离到1株J亚群禽白血病病毒(ALV-J),命名为ZH-08。利用ELISA抗原检测、PCR和间接免疫荧光试验对分离株进行鉴定,结果都呈阳性。依据ALV-J原型株HPRS-103前病毒全基因组序列设计并合成3对引物,采用分段扩增的方法完成了分离株的全基因组序列测定。结果显示该分离株基因组序列全长7 597 bp,与已公开的全基因组序列大小比较略有差异,但符合典型的复制完全型反转录病毒的基因组结构,基因序列中不含已知致癌基因。将该分离株的亚群特异性gp85基因序列与国内外各参考株相应序列进行相似性比较,发现ZH-08与YZ9901株相似性最高(93.7%)。基于gp85核苷酸序列的系统进化分析表明:ZH-08株与SD07LK1株的亲缘关系最近。本研究为该毒株的生物学特性以及致病机制研究奠定了基础。 相似文献