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1.
鸡三种肿瘤病快速鉴别诊断的研究初报 总被引：10，自引：0，他引：10
论文报告了利用聚合酶链反应（PCR）技术对马立克氏病（MD），淋巴白血病（LL）和网状内皮增生症（RE）三种鸡常见肿瘤病进行快速鉴别诊断的研究。初步确定了快速鉴别诊断的基本方法及PCR的反应条件，而且对从南宁、玉林、桂林等20个鸡场收集的肿瘤／可疑肿瘤组织共189份进行检测。结果MD的阳性率为79．09％，RE的为19．09％、LL的为7．27％，MD＋RE、MD＋LL二重阳性率以及三重阳性率分别为17．27％、7．27％和2．72％。本研究建立的肿瘤病快速鉴别诊断方法是敏感、特异和快速的。此外，还首次证实RE和LL在广西的存在与流行。 相似文献
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反转录聚合酶链反应和核酸探针检测禽呼肠孤病毒研究的概述 总被引：3，自引：0，他引：3
根据禽呼肠孤病毒(ARV)参考毒株S1133S1基因序列，设计合成4对引物而分别建立了RT-PCR、半套式PCR和一步法PCR三种用于检测ARV的方法。它们能检测到的最近ARVRNA量分别为1pg、1fg和0．16ngRNA；研制出禽呼肠孤病毒地高辛核酸探针，并建立了地高辛核酸探针检测ARV的方法，该核酸探针最低能检测到0．45ng的ARVRNA；对广西部分鸡场血清学调查表明，ARV平均阳性率为27％；对广西ARV野毒株进行了分离和鉴定，并对获得的2个地方分离株的S1基因片段进行测序及分析，两分离株与标准株S1133的同源性分别为98．5％和98．3％；用所建立的RT-PCR和地高辛核酸探针方法对用ARV人工感染鸡采集的各种样品的检测，并和传统病原分离方法进行比较，结果RT-PCR检出率为92．5％(222／240)，地高辛核酸探针检出率为74．2％(178／240)，病原分离鉴定方法检出率为55％(132／240)。用ARV强毒分别对免疫种鸡后代和无母源抗体或SPF的雏鸡进行攻毒试验，结果油苗免疫的种鸡后代对本病有很强的抵抗力，能抵抗ARV强毒的攻击，其平均保护率90．8％(109／120)。而无母源抗体或SPF的雏鸡攻毒后均100％(240／240)死亡。 相似文献
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鸡14种病毒病基因芯片的研究 总被引：4，自引：0，他引：4
应用PCR或RT—PCR方法分别获得鸡新城疫(ND)、禽流感(AI)、鸡传染性支气管炎(IB)、鸡传染性法氏囊病(IBD)、鸡传染性喉气管炎(ILT)、鸡马立克氏病(MD)、减蛋综合征(EDS-76)、禽网状内皮组织增生症(RE)、鸡脑脊髓炎(AE)、鸡传染性贫血症(CIA)、鸡病毒性关节炎(AVA)、包涵体肝炎(IBH)、禽白血病(AL)、鸡痘(FP)各一特异性片断，以这些片断为探针，采用接触式点样技术，制成低密度的DNA阵列芯片。自病料中提取核酸，通过生物标记技术，使样品核酸标记上生物素。当样品和预点在硝酸纤维素膜上的探针杂交后．通过显色技术，即可得出诊断结果。 相似文献
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用地高辛标记鸭圆环病毒（DuCV）全基因组克隆制成核酸探针,与鸭病毒性肝炎Ⅰ型病毒（DHV-Ⅰ）的cDNA、鸭瘟病毒（DPV）的DNA和DuCV的DNA进行斑点杂交以检测核酸探针的特异性,结果该探针只与DuCV的DNA杂交呈阳性。敏感性结果表明,该探针对DuCV的最低检出量约为5PgDuCV的DNA。应用该探针对从山东、江苏、四川等地采集的196只鸭病料,共980份脏器的组织DNA进行检测,结果个体阳性率为33．2％（65／196）,在各脏器中法氏囊和肝脏中DuCV检出率较高,检出率分别为52．3％和49．2％。同时对上述样品进行PCR检测,核酸探针检测与PCR检测2种方法的符合率为87．5％。结果表明,制备的DIG标记的核酸探针特异性强、敏感性高,适合对DuCV进行诊断和流行病学调查。 相似文献
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本研究把柑橘黄龙病（Citrus Huanglongbing，HLB）阳性核酸样品进行梯度稀释后，分别采用HLBp、CQULAP两组TaqMan水解探针法和以rpl基因为目的片段的SYBR Green染料法进行HLB实时荧光定量PCR(Quantitative Real-time PCR, qPCR)灵敏度检测研究，结果发现HLBp探针法可以从105倍稀释的阳性叶片核酸样品中检测到病原菌（病菌拷贝数约为60个），高于CQULAP探针法和SYBR Green染料法；将三种qPCR检测体系用于赣州安远县、寻乌县和赣县送检的60份田间样品检测，结果HLBp探针阳性率为86.66%，CQULAP探针阳性率为60%，以rpl基因为目的片段的SYBR Green染料法检测阳性率为68.33%。 相似文献
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产肠毒素性大肠杆菌重组菌PSLM004质粒经IaqI酶切,其840bp的片段用~(82)P-dATP标记后作为STI基因探针。菌落原位杂交表明,该探针可以和人源、猪源产STI的ETEC有阳性杂交反应,而不与不产STI的载体菌株pBR322、非致病性E.coli和其它肠道杆菌有杂交反应。用该探针对35株已知血清型的E.coli、65株从人腹泻分离的E.coli.和7株猪腹泻E.coli进行STI基因检测,分别有6株、7株和2株STI探针菌落杂交阳性,阳性率分别为17％(6╱35)、11％(7╱65)和28％(2/7)。乳鼠生物学试验比较研究表明,70％(11/16)的STI探针栓测阳性菌株其乳鼠生物学试验也为阳性。用该探针可以对产STI的ETEC进行临床检测和流行病学调查。 相似文献
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根据GenBank中已经发表的B亚型禽偏肺病毒F基因的保守序列设计并合成1对引物,利用RT—PCR扩增出1条与目的片段大小一致的725bp基因片段,回收、纯化PCR产物,用地高辛标记,制备出地高辛标记的aMPV核酸探针。特异性检测结果表明,该探针能与aMPV核酸发生特异性杂交,而与H9N2亚型AIV、NDV、IBV、ORT和E．coil的核酸杂交反应均为阴性;敏感性检测结果表明,该探针对aMPV的最低检出量为5Pg。应用制备的探针对山东省不同地区的605份商品肉鸡和122份商品肉鸭进行了核酸探针检测,阳性检出率分别为36．59％和34．51％。本试验制备的aMPV地高辛探针特异性强、敏感性好,对样品的检测结果表明山东省部分地区的商品肉鸡、肉鸭中普遍存在aMPV感染。 相似文献
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作者拟建立检测猪细小病毒(PPV)和猪圆环病毒2型(PCV2)的二重液相芯片(xMAP)检测方法,以满足出入境检疫和临床高通量检测的要求.依据GenBank中PPV结构蛋白VP2基因及PCV2全基因序列,设计PPV和PCV2特异性探针、引物及探针互补序列,将探针与磁性微球偶联,对下游引物和探针互补序列标记生物素,采用不对称性二重PCR扩增靶序列,将PCR产物与偶联好的探针进行杂交,用液相芯片仪进行检测.通过对检测条件优化,建立检测PPV、PCV2两种病毒的二重液相芯片技术,并对临床样本进行检测.建立的PPV和PCV2二重液相芯片检测方法,特异性良好,该方法对PPV和PCV2基因拷贝数检测下限分别为1.53×10 4、2.58×102;对56份临床样品检测表明,该方法与单项PCR检测结果符合率为100％.成功建立了PPV、PCV2二重xMAP检测方法,该检测技术可用于出入境检疫和兽医临床检验. 相似文献
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Three abortigenic Indian isolates of equine herpesvirus-1 (EHV-1) (Tohana, Hisar and Bikaner), along with two exotic abortigenic
isolates (AB4 and V592) and another EHV-1 isolate (Jind) obtained from a case of perinatal foal mortality, were studied for
variability. For this purpose, PCR and restriction endonuclease (RE) digestion techniques were used simultaneously as a DNA
fingerprinting system. Nine different regions of EHV-1 virus were amplified by PCR using primer pairs specific for the regions
and the products obtained from these regions were subsequently subjected to various restriction endonucleases to further assess
the variability in the number of RE sites as well as in their positions. No difference was observed in all the four abortigenic
isolates in terms of the size of different PCR products amplified by all the nine primer pairs, except for primer pairs ‘E’
and ‘C’. PCR products obtained with primer pair E revealed that Tohana and Bikaner isolates were most similar while Hisar
isolate was like V592 isolate. However, the PCR product obtained from Jind isolate had a size between the PCR products of
Hisar and Tohan/Bikaner isolates. The primer pair ‘C’ used to amplify the region between 1151 to 3679 in ‘Gene 1,2,3’ clearly
differentiated the EHV-1 isolate obtained from a case of perinatal foal mortality from isolates obtained from abortion cases.
This primer pair needs to be exploited more extensively for use as a potential marker for differentiating the EHV-1 isolates,
mainly the abortion cases from perinatal foal mortality ones. Restriction endonuclease studies done with PCR product of all
the isolates with various primer pairs did not reveal any changes in the position or number of RE sites present in the products
amplified, indicating no variation in different RE sites within the amplified PCR products. However, this study clarified
that all the Indian isolates belonged to the IP group of EHV-1. 相似文献
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bcl—2在鸡马立克氏病肿瘤中的表达 总被引：5，自引：1，他引：4
用来克亨SPF鸡复制马立克氏病（Marek‘s disease,MD)肿瘤模型，取肿瘤和相应正常组织，液氮保存。AGPC（Acid Guanidinium Thiocyanate-Phenol-Chloroform)法的取组织总RNA（TRNA），紫餐测定其浓度，甲醛变性胶电泳观察其纯度。将一个含鸡bcl-21.2kb片段的质粒（PTTCB1），经转化扩增，提取质粒DNA，限制性内切酶（RE）消化，回收纯化bcl-2片段，放射性α^32P标记，制成探针。通过Northern分子杂交检测MD肿瘤组织中bcl-2的表达，并与相应的正常组组织比较。结果：鸡MD肿瘤组织和相应正常组织中均有bcl-2的表达；MD肿瘤组织中bcl-2 的表达显著高于相应的正常组织。结合已进行的凋亡研究表明,bcl-2表达产物通过阻遏细胞凋亡促进MD淋巴瘤的形成。 相似文献
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用猪肾细胞 (PK- 15 )繁殖猪瘟病毒 (CSFV)分离株 HL - L Y,根据基因库已发表的 CSFV E2 基因序列 ,设计并合成一对引物 ,以 CSFV总 RNA为模板 ,通过 RT- PCR技术扩增出约 1.1kb的片段 ,即 E2 基因。将 RT- PCR产物先克隆到 p MD18- T载体上 ,构建了重组质粒 T- E2 ,再通过双酶切亚克隆到表达载体 p PIC9的多克隆位点 (MCS)上 ,经酶切、PCR鉴定和测序分析 ,表明均已成功获得了 CSFV E2 基因的重组体 p PIC9- E2 。将该测序结果与国内几个毒株 SHIMEN,C,C- V- L Z,GS- L T,GS- L X分别进行序列同源性比较 ,核苷酸同源性分别为 96 .5 1% ,95 .5 3% ,89.12 % ,78.6 6 % ,77.2 3% ;氨基酸同源性分别为 97.32 % ,95 .71% ,89.5 4 % ,83.16 % ,83.4 2 %。表明该毒株与国内标准毒株 SHIMEN株和 C株具有较高的同源性 相似文献
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1. A total of 3236 females from eight meat-type strains, half of which were vaccinated for Marek's disease (MD), and 11,193 Leghorn females from ten strains, all vaccinated for MD, were adventitiously exposed to MD and lymphoid leukosis (LL) viruses and observed to 392 and 497 d of age, respectively. 2. In the meat-type birds, vaccination reduced total mortality from 43-4% to 27-1% and mortality due to MD from 16-4% to 5-4% but did not affect mortality and LL (2-9% and 3-4%). 3. In the vaccinated Leghorns total mortality was 11%, including 2-1% from MD and 1-2% from LL. 4. Significant differences between strains of chickens were found in total mortality, as well as in MD and LL mortality. 5. Strain by vaccination interaction was observed in total rearing and adult mortality, as well as in the MD mortality of adult meat-type females. 6. Leghorn strains with higher rate of egg production and meat-type strains with lower growth rate to have better viability. 相似文献
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【目的】 在毕赤酵母中构建猫血清白蛋白（feline serum albumin,FSA）表达系统,并探索其最适的基本表达条件,为FSA的生产提供一种新方法。【方法】 在GenBank上获取FSA碱基序列,进行密码子优化并合成,将优化的密码子构建到载体pPIC9K上,经PCR和双酶切验证后通过电击转化将重组质粒FSA-pPIC9K转化毕赤酵母GS115,依次经MD平板和G418筛选后进行菌落PCR获得阳性菌株,随机选取阳性菌株经甲醇诱导表达96 h后,取表达上清液进行Western blotting验证。选取表达量较高的菌株分别在不同温度（24、26、28和30 ℃）、pH （4.0、5.0、6.0、7.0和8.0）和甲醇诱导量（其中1组为每24 h添加0.5%,其余4组为每12 h分别添加0.5%、1.0%、1.5%和2.0%）的条件下诱导表达96 h,取表达上清液进行Western blotting验证。【结果】 菌落PCR结果显示,获得2条大小分别约为2.3和2.2 kb的目的基因条带和毕赤酵母AOX1基因扩增条带,诱导表达96 h后取表达上清液进行Western blotting验证,在70 ku左右出现特异性条带。通过基本条件优化发现该蛋白在28 ℃,pH为6.0且每12 h添加0.5%的甲醇条件下表达量较高。【结论】 毕赤酵母GS115可稳定表达FSA。 相似文献
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Reticuloendotheliosis (RE) in captive greater prairie chickens (GPC, Tympanuchus cupido pinnatus) and Attwater's prairie chickens (APC, Tympanuchus cupido attwateri) was first reported in 1998. RE is caused by avian reticuloendotheliosis virus (REV), an oncogenic and immunosuppressive retrovirus infecting multiple species of wild and domestic birds. During August 2004 through May 2006 a captive population of prairie chickens was affected simultaneously with a neoplastic condition and also avian pox, the latter being detected in 7.4% (2 of 27) of all birds submitted for histopathology. A survey for REV was conducted in order to examine its possible role in mortality observed primarily in juvenile and adult specimens of prairie chickens. The investigative procedures included postmortem examinations, histopathology, molecular detection, and virus isolation. In total, 57 Attwater's prairie chickens and two greater prairie chickens were included in the study. REV infection was diagnosed using virus isolation or polymerase chain reaction (PCR) or both in 59.5% (28 of 47) of blood samples and/or tumors from suspect birds. Lymphosarcomas were detected in the tissues of 37% (10 of 27) of the birds submitted for histopathology. Such lymphosarcomas suggestive of RE represented the most frequent morphologic diagnosis on histopathology among 27 separate submissions of naturally dead prairie chickens. Overall, REV was detected or RE diagnosed in 34 of 59 prairie chickens (57.62%). The average death age of all birds diagnosed with lymphosarcomas on histopathology was 2.2 yr, ranging from <1 to 4 yr. Although deaths associated with neoplasia occurred in males and females in equal proportions based on submissions, overall more males were diagnosed as REV infected or RE affected (16 males vs. 7 females, and 11 birds of undetermined gender). Reticuloendotheliosis virus was confirmed as a significant cause of mortality in captive prairie chickens. 相似文献
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Lemiere S Fernández R Pritchard N Cruz-Coy J Rojo F Wong SY Saint-Gerand AL Gauthier JC Perozo F 《Avian diseases》2011,55(4):642-649
Hatchery vaccination protocols in day-old chicks are designed to provide early priming and protection against several poultry diseases including, but not limited to, Marek's disease (MD), infectious bursal disease (IBD), and Newcastle disease (ND). The constraint of concomitant administration of live MD and IBD vaccines plus ND inactivated oil-adjuvanted vaccines (IOAVs) requires improvements in vaccine technology. Single-needle concomitant subcutaneous (SC) application of IBD/MDV and killed NDV vaccine and the use of viral vectors for expression of immunogenic proteins are a current trend in the industry. The objective of this work was to assess the compatibility of a turkey herpesvirus (HVT)-infectious bursal disease (vHVT-IBD) vector vaccine applied simultaneously with IOAV and to evaluate the consequences for vaccine intake, the need for additional immunizations with the respective vaccines, and protection. Five separate trials were performed using double- and/or single-needle injectors. The levels and persistence of vaccine intake, serologic response, vHVT-IBD virus combination with the MD Rispens strain, and/or live NDV vaccination were also assessed. Histopathology and PCR at injection sites showed adequate vaccine intake detected up to 44 days postvaccination. Serologic evidence of vaccine priming was observed, and all vaccinated groups differed (P < 0.05) from the control at different time points. MD, NDV, and IBD protection results after concomitant double-shot single-needle vaccination were near 85%, 95%, and 100%, respectively. Taken together the results indicate no deleterious effects on the efficacy of the vHVT-IBD vaccine monitored by vaccine intake, serologic and challenge results, and combinations after concomitant live/killed vaccination, suggesting the suitability of its use in hatchery vaccination. All types of injectors used as well as injection techniques, vaccines injected separately or together, gave the same results. 相似文献
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Gomes LI Rocha MA Souza JG Costa EA Barbosa-Stancioli EF 《Veterinary research communications》2003,27(6):495-504
Bovine herpesvirus type 5 (BoHV-5), which is potentially neuropathogenic, was detected in clinical samples of bovine semen, both directly and after isolation in cell culture, using a nested PCR system for amplifying the US4 gene. Nucleotide sequences generated from the amplicons were analysed and deposited at GenBank (NCBI, Bethesda, MD, USA) under the accession numbers AF298174 and AF330157. Alignment of these sequences and previously deposited sequences of BoHV-1 and BoHV-5 showed 82% and 98% similarity, respectively. The bulls, which were maintained at an artificial insemination centre, had presented no clinical signs, indicating that bovine semen should be screened for BoHV-5 to prevent transmission of the virus. 相似文献
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A longitudinal field study of mortality and Marek's disease in Norwegian and imported White Leghorns
B. T. Heier J. Jarp M. I. Kaldhusdal G. Schaller I. B. Forus 《Preventive veterinary medicine》1999,40(3-4):207-219
High mortality during the first part of the laying period was observed in Norwegian White Leghorns during the period 1988–1992. A longitudinal field study with repeated measurement of cumulative mortality was undertaken in the period from January 1994 to January 1996 to investigate (1) the mortality and susceptibility to Marek's disease (MD) in the Norwegian strain (NB41) compared to two imported layers, (2) the effect of MD on the total cumulative mortality in the period from 16 to 32 weeks of age in White Leghorn flocks, and (3) the effect of MD as judged by repeated measurement of cumulative mortality in the same period. All five layer hatcheries and 67% of the pullet-rearing farms in Norway participated in the field study. The egg-production farms were sampled by convenience. Recordings for the whole period were completed for 169 flocks in 101 farms. The statistical analyses were performed using both a general fixed-effects linear model and a mixed model with repeated measurements, with total flock-level cumulative mortality and flock-level cumulative mortality in four-week intervals as outcome variables, respectively.
The overall cumulative flock-level incidence of MD was 12% (24% and 8% in NB41 and Lohmann White, respectively). MD was not recorded in any of the Shaver White flocks. A significant difference (p < 0.001) was found in (1) total cumulative mortality: 8.2% in the NB41 and 5.0% in the imported layers, and (2) ‘interval-specific' cumulative mortality: 0.36% in the NB41 and 0.15% in the imported birds. A strong relationship was also demonstrated between MD and repeated measurements of ‘interval-specific' cumulative mortality (p < 0.001) but not when cumulative mortality was used as an overall measure for the whole laying period (p = 0.11). The results from the repeated-measures analysis also indicated a stronger effect of MD on flock-level ‘interval-specific' cumulative mortality in the NB41 than in the imported hens. The different cumulative mortality and susceptibility to MD observed in the NB41, compared to the imported hens, shows that the farmers will be able to reduce their losses by replacing the NB41 strain with one of the imported strains. 相似文献