共查询到20条相似文献，搜索用时 15 毫秒
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鸡呼吸道传染病基因芯片诊断方法的建立 总被引：4，自引：0，他引：4
随着养禽业集约化程度的不断提高,鸡呼吸系统疾病的发生呈逐年上升趋势.而且大多数的呼吸道疾病不是单一病原感染,而是多种病原混合感染,从临床上难以及时鉴别诊断,延误防治时机,从而给养禽业造成巨大的经济损失.因此,禽呼吸道疾病已经成为生产和研究中的一类重要疾病.目前,禽呼吸系统疾病的诊断方法主要有病毒分离、血凝（HA）、血凝抑制（HI）、琼脂扩散、ELISA、PCR等,但是传统的检测方法灵敏度较低,而且当病原发生变异或感染禽处在潜伏期时会造成误诊.PCR方法每次只能检测一种病毒,费时费力.采用基因芯片的方法可以快速、准确地同步检测多种病原体,且需要样品量少、灵敏、特异、快速、费用低廉,将生物芯片技术用于禽呼吸道疾病诊断意义重大. 相似文献
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Wang J O'Keefe J Orr D Loth L Banks M Wakeley P West D Card R Ibata G Van Maanen K Thoren P Isaksson M Kerkhofs P 《Veterinary microbiology》2008,126(1-3):11-19
Six laboratories participated in a ring trial to evaluate the reliability of a real-time PCR assay for the detection of bovine herpesvirus 1 (BoHV-1) from extended bovine semen. Sets of coded samples were prepared and distributed to each of the laboratories. The sample panel contained semen from naturally and artificially infected bulls, serial dilutions of positive semen with negative semen, semen from uninfected seronegative bulls, negative semen spiked with virus, as well as serial dilutions of reference virus. The samples were tested using a previously validated real-time PCR assay for the detection of BoHV-1 in each participating laboratory. The PCR tests were conducted with four different real-time PCR amplification platforms, including RotorGene 3000, Stratagene MX 3000/4000, ABI 7900, and Roche LightCycler 2.0. Virus isolation using one set of samples was performed in one laboratory. The results of the laboratories were compared with one another, and with those of virus isolation. It was found that the sensitivity and specificity of the real-time PCR test was greater than those of virus isolation (82.7% versus 53.6% and 93.6% versus 84.6%, respectively). A high level of agreement on PCR testing results between the laboratories was achieved (kappa value 0.59-0.95). The results of this study indicate that the real-time PCR assay is suitable for the detection of BoHV-1 in extended semen, and would be a good substitute for the slow and laborious virus isolation, for the screening testing at artificial insemination centres and for international trade. 相似文献
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Tell LA Leutenegger CM Larsen RS Agnew DW Keener L Needham ML Rideout BA 《Avian diseases》2003,47(4):1406-1415
Diagnosis of avian mycobacteriosis, caused by Mycobacterium genavense or species belonging to the Mycobacterium avium complex (MAC), is problematic. Polymerase chain reaction (PCR) offers rapid and sensitive detection of minute quantities of DNA, and conventional protocols have been used for evaluating avian specimens. The recent development of real-time PCR offers several advantages over conventional PCR. In attempts to improve diagnosing avian mycobacteriosis, a real-time TaqMan PCR assay was developed targeting the 65-kD heat shock protein gene of M. genavense and MAC spp. Nineteen reference isolates, 16 clinical isolates, and 32 avian tissue samples were used to evaluate the assay. When sufficient amplicons were produced, the species of mycobacteria was determined by standard sequencing of TaqMan PCR products and compared with results from commercial mycobacteriology laboratories and/or standard sequencing of conventional PCR products. The TaqMan PCR detected DNA from reference isolates of M. genavense, MAC spp., and Mycobacterium tuberculosis complex spp. Of the clinical isolates, the TaqMan PCR detected DNA from 10 of 12 Mycobacterium avium avium isolates and two of three Mycobacterium avium intracellulare isolates. For the tissue samples, the TaqMan PCR amplified DNA in six of nine samples that were identified by sequencing of conventional PCR products and/or by commercial mycobacteriology laboratories as being MAC spp. positive and three of four samples that were positive for M. genavense. There was some disagreement between speciation results from the TaqMan PCR and those from commercial mycobacteriology laboratories or conventional PCR or both. This disagreement was suspected to be because of relatively small numbers of base pairs in the TaqMan PCR products. The TaqMan PCR may provide a useful tool for evaluating clinical samples for DNA from mycobacteria species that most commonly infect birds; however, further refinement is needed in order to improve sensitivity and provide more accurate speciation. 相似文献
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选取H5亚型禽流感病毒血凝素(Hemagglutinin,HA)基因保守序列,使用Primer Express 2.0软件设计出特异性引物和TaqMan MGB探针,利用实时荧光PCR技术来定量检测禽流感病毒。使用含有选定检测序列的RNA标准品做标准曲线,结果表明该方法的灵敏度为10拷贝/反应,标准曲线的相关系数为0.998003,对H9亚型禽流感病毒和其他禽病病毒无交叉反应,特异性好、重复性佳。为禽流感病毒检测提供了一种特异、敏感、快速的定量检测方法。 相似文献
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H5N1亚型高致病性禽流感病毒A/duck/HuBei/49/05株反向基因操作系统的建立 总被引：1，自引：0，他引：1
水禽是禽流感病毒的天然储存库,为了研究高致病性禽流感病毒对水禽致病性的分子基础,研究构建了DKHB/49/05的8质粒反向遗传操作系统。用Trizol从含A/duck/HuBei/49/05株病毒的鸡胚尿囊液中提取总RNA,用RT-PCR扩增PB2、PB1、PA、HA、NP、NA、M、NS基因片段,将其分别克隆至pBD载体中。利用8质粒反向遗传操作系统,将重组的pBD质粒共转染293T细胞,成功拯救了该病毒,并命名为R-DKHB。 相似文献
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Classical swine fever virus: a second ring test to evaluate RT-PCR detection methods 总被引：5，自引：0，他引：5
Paton DJ McGoldrick A Bensaude E Belak S Mittelholzer C Koenen F Vanderhallen H Greiser-Wilke I Scheibner H Stadejek T Hofmann M Thuer B 《Veterinary microbiology》2000,77(1-2):71-81
Six laboratories participated in a study to compare the sensitivity and specificity of RT-PCR tests for the detection of classical swine fever virus (CSFV). Sets of coded samples were prepared by serial dilution of positive samples and then distributed to each of the laboratories. One set comprised 25 samples of random primed cDNA, synthesised from viral RNA representative of different pestiviruses. The other set comprised samples of blood and serum obtained from virus-free or CSFV-infected pigs. Each laboratory tested the samples using PCR/RT-PCR according to a set of standardised protocols that specified the exact conditions and requirements for inclusion of control samples. Two types of test were evaluated. One amplified a part of the 5'-non coding region of the pestivirus genome by means of a closed, one-tube RT-nested PCR. The other amplified a part of the NS5B gene using non-nested RT-PCR. The results of the laboratories were compared with one another, and with those obtained earlier when similar samples were tested by the same laboratories using non-standardised methods [Paton et al., Classical swine fever virus: a ring test to evaluate RT-PCR detection methods, Vet. Microbiol., in press]. Standardisation of the protocols resulted in a more consistent test sensitivity. Three laboratories avoided significant false positive results. Others that did not, could nevertheless recognise that test specificity was inadequate from the results obtained with the control samples. Minimum requirements for the inclusion of adequate controls and periodic proficiency testing are proposed. 相似文献
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根据禽呼肠孤病毒（ARV）、禽流感病毒（AIV）、新城疫病毒（NDV）和鸡传染性支气管炎病毒（IBV）的基因保守区设计了4对特异性引物,建立了针对四种病毒的多重PCR检测体系,并对该体系进行了条件优化及特异性、敏感性试验。该体系扩增的四种病毒基因片断大小分别为199bp（ARV）、264bp（AIV）、362bp（NDV）和459bp（IBV）,且特异性、敏感性良好,能够检出1pgAIV和10PgARV、NDV、IBV的RNA。临床应用结果证明,该体系具有很高的实用价值和应用前景。 相似文献
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利用基因克隆和体外转录技术,制备A型禽流感病毒通用核酸检测阳性标准样品。根据禽流感基质蛋白基因M的序列,设计全长开放阅读框的克隆引物,RT-PCR获得相应片段,连接至pGEM-T载体,测序后采用体外转录方法制备RNA纯品,初步定量稀释后,混合分装作为核酸标准样品候选物。采用实时荧光定量RT-PCR方法进行均匀性和稳定性检验,并委托外部实验室采用外标实时定量RT-PCR方法对转录的RNA片段进行定值。通过绘制荧光定量标准扩增曲线和协作标定的方式,计算标准物质含量（拷贝）,并进行不确定度的估算。均一性结果显示瓶间差异小于5%,稳定性试验表明室温20~25℃（相对湿度20%~50%）14d,2~8℃3个月以及-20℃保存1年的含量均无明显变化。核酸标准物质定值为（3.120±0.345）×10^8拷贝数,可用作禽流感病毒通用核酸检测的标准质控品。 相似文献
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FENG Bin XIE Zhi-xun ZHANG Yan-fang HUANG Jiao-ling WANG Sheng FAN Qing HUANG Li XIE Li-ji ZENG Ting-ting LUO Si-si DENG Xian-wen XIE Zhi-qin LIU Jia-bo SONG Zhong-bao LIN Er-ke 《中国畜牧兽医》2017,44(5):1295-1301
In order to set up and optimize a semi-nested PCR for rapid detection of chicken parvovirus (ChPV), three specific primers were designed according to conserved sequences of NS 1 gene of ChPV. The specificity and sensitivity of ChPV semi-nested PCR were tested, and the assay was applied to detect 48 clinical samples. The specificity and sensitivity tests showed that this semi-nested PCR was only sensitive to ChPV for amplifying specific band of 186 bp and it could detect 5.62 fg/μL of ChPV DNA, without any sensitivity to other viruses, such as Newcastle disease virus, H9 subtype avian influenza virus, Marek's disease virus, infectious laryngotracheitis virus and infectious bronchitis virus. 48 chicken samples were detected and the positive rate was 16.67% (8/48). The results of our study demonstrated that the optimized semi-nested PCR could be a method that was suitable for clinical detection of ChPV. 相似文献
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为建立一种快速、特异、灵敏的检测鸡细小病毒（chicken parvovirus,ChPV）的方法,根据ChPV的保守基因NS1设计了3条特异性引物,建立并优化了能快速检测ChPV的半巢式PCR方法,对其进行特异性和敏感性试验,并用所建立的方法对48份临床样品进行了检测。特异性和敏感性试验结果显示,建立的半巢式PCR只对ChPV敏感,扩增产物为186 bp的特异性条带;其最低能检测到5.62 fg/μL的ChPV DNA;而对鸡新城疫病毒、H9亚型禽流感病毒、马立克氏病病毒、鸡传染性喉气管炎病毒、鸡传染性支气管炎病毒不敏感。临床检测结果显示,同时对48份临床样品进行检测,检出率为16.67%（8/48）,提示广西区内鸡群存在ChPV感染。本研究建立的ChPV半巢式PCR方法适用于ChPV的临床检测。 相似文献
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旨在调查野鸟中存在的禽流感病毒,并为禽流感的防制提供依据。采集新疆艾比湖自然保护区野鸟的粪便棉拭子进行禽流感病毒的分离与鉴定,将PCR产物进行纯化后用T载体连接并转化到DH5α细胞中;菌液PCR鉴定后将目的菌液送至上海生工测序并比对测序结果,在NCBI上进行基因序列分析。结果显示,从新疆艾比湖自然保护区野鸟的粪便棉拭子分离得到1株H1N2亚型禽流感病毒;经序列分析得知,该毒株为1株低致病性禽流感病毒,且存在与多种亚型病毒的重配现象。 相似文献
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从福州市某活禽市场采集的鸡泄殖腔棉拭子样品中分离出1株病毒,经血凝抑制试验(HI)和聚合酶链反应(PCR)方法鉴定为H9N2亚型禽流感病毒。在GenBank基因库中对该病毒株的3个基因片段的测序结果进行BLAST比对分析表明,3个基因片段均属于H9N2亚型禽流感病毒的基因。HA基因遗传进化分析表明,该病毒分离株与代表株DK/HK/Y280/97处于同一分支,与上海的鸡源分离株A/chicken/Shanghai/06/2015(H9N2)同源性最高,同源性为99.5%。 相似文献