共查询到19条相似文献，搜索用时 62 毫秒
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为建立敏感、特异的评价猪瘟兔化弱毒(HCLV)疫苗中病毒含量的实时荧光定量RT-PCR方法,参照中国猪瘟石门株兔化弱毒全长序列,在猪瘟兔化弱毒活疫苗基因组5'非编码区设计1对标准品引物、1对特异性引物和1条探针,建立检测猪瘟兔化弱毒活疫苗病毒含量的实时荧光定量RT-PCR方法.该方法检测的敏感度达1.20×105拷贝/mL;对猪繁殖与呼吸综合征、猪乙型脑炎、仔猪副伤寒和猪伪狂犬病4种活疫苗基因组扩增结果均为阴性;重复性试验结果显示,批内变异系数为0.29%～0.39%,批间变异系数为0.32%～0.61%.应用此方法对6个不同厂家生产的7种猪瘟兔化弱毒活疫苗中病毒含量进行了检测,发现不同厂家生产的疫苗中病毒含量存在较大差异.结果表明,建立的猪瘟兔化弱毒疫苗实时荧光定量RT-PCR方法能特异地检测疫苗病毒含量,可用于初步评价猪瘟兔化弱毒疫苗抗原含量. 相似文献
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SYBR Green Ⅰ实时荧光定量RT-PCR检测猪瘟疫苗病毒含量 总被引：1，自引：0，他引：1
为建立一种能够快速定量检测猪瘟兔化弱毒疫苗病毒含量的实时荧光定量RT-PCR方法。对GenBank登录的25株猪瘟病毒强毒株和兔化弱毒疫苗株基因组全序列进行比较分析,在其高度保守的5′端非编码区设计1对针对猪瘟兔化弱毒疫苗的特异性引物,扩增片段为245 bp,且不与牛病毒性腹泻病毒以及其他猪源病毒发生非特异性反应。应用实时荧光定量RT-PCR法对16份猪瘟脾淋苗和细胞苗进行定量检测,结果表明,102拷贝/μL的总RNA即能得到特异性扩增,在107~102拷贝/μL线性范围内有良好的扩增曲线,并与兔体定型热反应有良好的相关性。该法具有敏感性、特异性、重复性好等优点,可望取代传统的兔热法用于猪瘟疫苗生产过程中的效价测定及指导疫苗的配制,也为猪瘟病毒分子生物学研究提供一种新的有效工具。 相似文献
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荧光定量RT-PCR方法与兔体定型热试验对于检测猪瘟兔化弱毒疫苗的平行关系 总被引：1，自引：1，他引：0
为探讨荧光定量RT-PCR技术与传统兔体定型热试验对于检测猪瘟兔化弱毒疫苗的平行关系,本研究采用荧光定量RT-PCR方法与兔体定型热试验对5份猪瘟兔化弱毒细胞苗成品和6份半成品样品进行平行检测.结果表明,两种检测方法存在正相关性,最低105个病毒拷贝可以使家兔产生定型热反应,最低103个病毒拷贝可以使家兔产生轻热反应.荧光定量RT-PCR检测方法的检测过程仅需3.5h,大大缩短了猪瘟疫苗的检测时间,该方法可以补充传统的兔体定型热反应用于猪瘟兔化弱毒疫苗半成品与成品的检验. 相似文献
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为了更准确地检测猪瘟疫苗中的病毒含量,为猪瘟免疫预防工作提供科学参考,试验设计针对CSFV E2基因的引物,利用RT-PCR方法扩增出目的片段,运用检测猪瘟病毒的SYBR Green-Ⅱ荧光定量PCR方法对来自不同生产厂家的6种猪瘟疫苗的病毒含量进行定量检测。结果:不同厂家生产的猪瘟兔化弱毒疫苗存在效价差异,6种疫苗中的病毒含量由高到低为ADFCEB,分别为4.52×10~4、2.36×10~4、2.02×10~4、1.70×10~4、1.08×10~4、6.18×10~3 copies/μL。 相似文献
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为了建立一种快速、特异、灵敏的检测牛病毒性腹泻病毒(BVDV)Taq Man实时荧光定量RT-PCR的方法,根据NCBI GenBank上已公布的BVDV、猪瘟病毒(CSFV)核酸序列进行比对,利用Oligo 6.71软件设计一对引物及一条探针,建立了检测BVDV的Taq Man实时荧光定量RT-PCR方法。通过对该方法的特异性、重复性及其敏感性进行相关试验,结果表明该方法检测出BVDV标准毒株Oregon C_(24)V为阳性,猪圆环病毒2型、猪伪狂犬病病毒、猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪繁殖与呼吸综合征病毒和CSFV的检测均为阴性。对BVDV标准毒株最低检测量达到10~(-2.5) TCID_(50)。该方法检测同一样品重复进行8次检测,结果均一致,表明方法的重复性较好。应用该方法对6批猪瘟疫苗专用血清进行BVDV检测,阳性率为16.7%;猪瘟弱毒苗中未检出BVDV。建立的BVDV Taq Man实时荧光定量RT-PCR方法为生产无BVDV污染的猪瘟苗提供了有力的保障。 相似文献
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根据GenBank中的猪瘟病毒强毒和弱毒株基因组序列设计了1对针对猪瘟病毒的通用引物和2条分别针对猪瘟病毒强毒和猪瘟兔化弱毒疫苗株的特异性TaqMan水解探针，建立了一种能区分猪瘟病毒强毒和兔化弱毒疫苗株的复合实时荧光定量RT—PCR检测方法。结果显示，该方法能将我国大陆流行的不同基因亚群的猪瘟病毒强毒株与猪瘟兔化弱毒疫苗株完全区分开来，而不与其他猪源病毒发生非特异反应，分别可检测到初始模板中41．8和81．5个拷贝的病毒RNA，与已建立的复合RT-套式PCR的敏感性相近，两种方法对152份样品检测的符合率达96．9％～100％。通过对8份猪瘟兔化细胞疫苗效价的检测，证实本方法与兔体反应热测定法有一定的相关性，可用于猪瘟病毒强毒株和兔化弱毒疫苗的定量和鉴别检测。 相似文献
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为了解猪瘟病毒感染仔猪免疫猪瘟疫苗后带毒情况,并比较实验室几种猪瘟抗原检测方法的适用性,采用(CSFV)RT-nPCR、猪瘟兔化弱毒疫苗荧光定量RT-PCR(HCLV-FQ-PCR)和CSFV实时荧光定量RT-PCR(CSFV-FQ-PCR)3种检测方法对田间感染CSFV仔猪疫苗免疫前后带毒情况进行定期跟踪检测.结果显示:本实验室建立的CSFV-FQ-PCR灵敏度高于CSFV-RT-nPCR;猪瘟疫苗免疫48 d后,采用HCLV-FQ-PCR方法检测不到血液中的HCLV;猪瘟病毒感染猪免疫疫苗后仍存在持续带毒现象,因此对猪瘟病毒感染猪必须彻底淘汰. 相似文献
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姚俊 《中国动物传染病学报》2012,20(6):68-76
为了建立一种既能检测野毒,又能检测疫苗毒的猪瘟病毒TaqMan实时荧光定量PCR方法,经过对GenBank中所有瘟病毒属成员高度保守的5’端非翻译区序列比对分析后,设计出1对TaqMan Real-time RT-PCR引物、1条TaqMan探针和3条RNA标准品制备引物。猪瘟病毒石门毒株经RNA标准品制备引物RT-PCR扩增后,再经T7 RNA聚合酶体外转录制备包含检测目的片断序列的猪瘟病毒RNA标准品。通过最佳引物、探针浓度的筛选及反应条件的优化,建立了猪瘟病毒TaqMan实时荧光定量PCR检测方法。该方法批内及批间重复试验变异系数均低于2%,特异性试验仅能检测出猪瘟强毒及疫苗毒株,最低浓度检测极限为1 X 102 copies/μL,上机检测时间少于60 min,建立的标准曲线斜率(Slope)为:-3.97,截距(Intercept)为:47.41,相关系数(R2)为:0.999779。运用该方法对3份猪瘟临床组织样品及6家企业生产的5种细胞苗及2种脾淋苗进行定量检测,结果提示:临床病料含有的病毒拷贝数差异不大,而疫苗产品每头份含有的病毒拷贝数差异较大。所建立的方法具有特异、快速、灵敏、可重复性和线性关系好的特点,不仅适合于猪瘟临床样品的早期检测,也适用于疫苗生产过程中的质控及疫苗制品的效价评估。 相似文献
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为进一步验证猪瘟兔化弱毒(HCLV)疫苗荧光定量RT-PCR(qRT-PCR)与兔体定型热试验之间存在正相关性,本研究利用qRT-PCR方法对278批次HCLV疫苗进行检测,分别检测其疫苗含量及牛病毒性腹泻病毒(BVDV)污染情况。结果表明,278批次猪瘟疫苗中有66批(24%)疫苗含量达不到规程标准,有42批(15%)疫苗存在BVDV污染;从所检疫苗中抽取6份qRT-PCR检测HCLV含量较低的疫苗,采用兔体定型热试验进行验证,两种方法结果吻合,表明qRT-PCR方法可以作为评价猪瘟疫苗质量的一种备选方法。 相似文献
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根据GenBank上已发表的猪瘟病毒(CSFV)和牛病毒性腹泻病毒(BVDV)的全基因序列,进行对比分析,分别设计合成两对能特异性扩增CSFV、BVDV的引物。经过条件优化后,建立了检测CSFV和BVDV的双重RT-PCR方法,扩增两种病毒的片段,大小分别为938、650 bp。应用该方法对11批牛睾丸细胞、7批胎牛血清、60个批次的猪瘟细胞苗、10份全血样及10份组织样进行检测。通过试验证明,所建立的方法具有良好的特异性和敏感性,为防止猪瘟细胞苗的污染及进行CSFV和BVDV鉴别诊断提供了有效方法。 相似文献
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通过向无外源病毒污染的鸡痘病毒活疫苗中添加不同剂量的禽网状内皮组织增生症病毒(REV),然后用间接免疫荧光法(IFA)进行检测,确定了该IFA方法的最低检出量为每500羽份疫苗中污染20 TCID_(50)的REV。使用该方法对国内16家企业生产的60批鸡痘病毒活疫苗进行了检验,结果显示2个企业生产的4批疫苗REV检测为阳性。随机选取5批IFA检测阴性样品和4批IFA检测阳性样品,按鸡检查法进行外源病毒检验,结果两种方法对REV污染的检测结果的符合率为100%。 相似文献
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Subclinical low-prevalent Aujeszky's disease (AD) serological test reactors test reactors are defined as those few swine within a qualified AD virus (ADV)-negative herd that have antibodies to wild type virus. However, clinical signs of the associated diease are not observed in these putatively infected swine or elsewhere in the herd. Twelve such animals, including 7 previously vaccinated with a genetically modified ADV, were identified in Illinois (USA) during a 2.5 year period. The humoral immune responses of the 5 nonvaccinated swine were assessed by an enhanced virus neutralization test and a radioimmunoassay. Anti-ADV antibodies were determined to be present in the serum from 4 of these swine. Attempts to isolate ADV by in vitro and in vivo inculations of cell cultures and weanling mice, respectively, of tonsillar and trigeminal nerve ganglionic tissue preparations from each animal (vaccinated and nonvaccinated) were unsuccessful. Tonsillar and trigeminal nerve ganglionic tissues of each animal were screened for the presecence of wild type and/or vaccine viral genomes by a soluble polymerase chain reaction (PCR) coupled with Southern hybridization. Unique PCR primers were used distinguish between wild type and vaccine viral DNAs. Additional PCR procedure, which amplifiers a portion of the essential and highly conserved viral gp50 gene, also was employed in an effort to detect viral genomes. Wild type viral DNA was found in the tissues from at least 5 of the vaccinated and 3 of the nonvaccinated swine. These results indicate that such animals should be considered as being infected with ADV. Further, these findings emphasize the need to develope highly specific and sensitive antemortem testing methods for accurate assessment of ADV infection in herds containing such subclinical, yet serologically positive, swine. 相似文献
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为了解某集约化猪场伪狂犬病病毒（PRV）的感染情况,清除感染猪只,净化伪狂犬病,提高猪群的健康水平.采用PRV gE抗体ELISA检测试剂盒,对该场免疫了猪伪狂犬病基因缺失疫苗的猪群进行3次野毒感染检测;应用荧光PCR方法,对临床发病的仔猪进行PRV病原检测.经过动态监测、加强综合防控和逐步淘汰措施,该猪场的PRV野毒感染率从35.29％逐步降低至0％,猪场自繁的仔猪存活率高,无PRV感染,净化效果良好. 相似文献
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Zhao JJ Cheng D Li N Sun Y Shi Z Zhu QH Tu C Tong GZ Qiu HJ 《Veterinary microbiology》2008,126(1-3):1-10
Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), one of OIE listed diseases. Most of the currently available detection methods do not allow discrimination between wild-type CSF viruses and the vaccine strains. This study was designed to develop a multiplex real-time RT-PCR for the quantitative and differential detection of wild-type viruses and C-strain vaccine widely used in China. CSFV specific primers and two differently labeled TaqMan probes for the differentiation of wild-type viruses from C-strain vaccine were designed in the 5'-untranslated region of the viral genome of CSFV. The two TaqMan probes specifically hybridize wild-type viruses of different subgroups and C-strain vaccine, respectively, in the multiplex real-time RT-PCR, with no cross-reaction to a number of non-CSFV porcine viruses. The sensitivity of the assay for detecting wild-type and C-strain-type vaccine viruses was determined to be 41.8 and 81.5copies/microL viral RNA, respectively. Completely correct differentiation of wild-type viruses from C-strain vaccine was achieved when testing reference strains and characterized field isolates of CSFV in China. The multiplex real-time RT-PCR was able to detect the viral RNA in the whole blood samples of experimentally infected pigs as early as 2 days post-infection, 3 to 4 days prior to the onset of clinical signs in co-housed pigs. The agreements between the multiplex real-time RT-PCR and a multiplex RT-nested PCR for detection of wild-type and C-strain-type viruses were 96.9% and 100%, respectively, when detecting 106 different field samples. There is a positive correlation between the titers of C-strain vaccines titrated in rabbits and RNA copies quantitated by the multiplex real-time RT-PCR. The novel assay described here is rapid and sensitive, and is useful for differentiating field strains and C-strain of CSFV in China. 相似文献
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Wenjun Ma Kelly M Lager Juergen A Richt William C Stoffregen Fanghong Zhou Kyoung-Jin Yoon 《Journal of veterinary diagnostic investigation》2008,20(4):440-447
The successful eradication of pseudorabies in U.S. domestic swine was accomplished through the use of glycoprotein E (gE) deleted modified live virus vaccines and an accompanying gE differential enzyme-linked immunosorbent assay (ELISA). Yet, pseudorabies virus (PRV) was established in feral swine in the United States, becoming a potential reservoir of PRV for infection of domestic swine and other native wildlife. A critical need for the current PRV surveillance program in the United States is the rapid detection of PRV infection. For this reason, a set of 2 real-time polymerase chain reaction (PCR) assays by using TaqMan chemistry was developed and evaluated for their capability in the detection and differentiation of field and vaccine strains of PRV. PCR primers and probes were designed for gB and gE genes of PRV, respectively. The newly developed PRV-specific real-time PCR assays could detect all wild-type PRV isolates from diagnostic submissions and differentiate them from vaccine strains. The analytical sensitivity of the assays was approximately 0.1 plaque-forming units per reaction. The assays were highly specific for PRV, because no positive results were obtained from testing other common swine viral pathogens and other animal herpesviruses. The results of testing samples from domestic and feral swine and from bovine showed that the real-time PCR assays are more sensitive than gel-based PCR. These results demonstrated the potential application of the developed real-time PCR assays as a differential test for rapid and specific detection of PRV in domestic and feral swine, as well as nonporcine species that can be infected with PRV and serve as carriers. 相似文献
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在动物疫苗研制过程中,应用巢式PCR方法,设计两套引物,扩增支原体16S与23SrRNA基因间隔区序列,可以检测造成细胞污染的常见支原体种类。本试验应用该方法检测三批样品和阴性对照均无特异性目标条带,即三批疫苗样品支原体检测均为阴性。阳性对照样品在200-400bp之间出现特异性目标条带,实验表明所建立的巢式PCIL方法是一种快捷、灵敏、准确的检测方法,可以用于检测动物疫苗细胞培养物中支原体污染。 相似文献