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1.
为建立一种能够区分猪瘟病毒(CSFV)强毒与弱毒疫苗C株的SYBR Green Ⅰ荧光定量RT-PCR结合熔解曲线分析方法,本研究对GenBank中登录的25株CSFV强毒株和兔化弱毒疫苗C株全基因组序列进行比较分析,设计一对共用下游引物以及分别针对CSFV强毒株与弱毒疫苗的特异性上游引物,其Tm值分别为84.5±0.5℃和88.5±0.5℃,熔解曲线分析显示为单特异峰.检测结果显示本实验建立的鉴别CSFV强毒感染与弱毒疫苗的荧光定量RT-PCR结合熔解曲线分析方法特异性强,对其他相关病毒无特异性扩增;敏感性高,最低检出量为5×RID50的细胞疫苗基因组拷贝;重复性好;并且扩增效率高、线性范围广、检测时间短,可对免疫猪群中CSFV强毒感染做出快速准确的鉴别检测,为有效防制猪瘟提供依据. 相似文献
2.
猪瘟病毒野毒株RT-LAMP可视化检测方法的建立 总被引：2，自引：0，他引：2
本研究旨在建立一种可视化检测猪瘟病毒(CSFV)野毒株的反转录.环介导等温扩增方法(RT-LAMP).根据CSFV的NS5B基因序列设计一套RT-LAMP引物,以样品的cDNA为模板,利用Bsf DNA聚合酶,在62℃恒温条件下进行扩增,扩增产物中加入sYBR Green I染料直接或在紫外光下观察判定扩增结果.该方法可检测出不同基因型的CSFV厂野毒株,其检出极限为2.5 TCID_(50)的CSFV,与实时荧光定量RT.PCR方法的敏感性相当;特异性试验表明,该方法对猪瘟免化弱毒疫苗株(HCLV)、牛病毒性腹泻病毒以及其它常见猪源病毒均无扩增反应;通过对126份不同样品进行检测比较,该方法与实时荧光定量RT-LAMP检测方法的符合率达100%.与引物.探针能量转移PCR方法的符合率为98.4%.该方法无需特殊仪器,是一种适用于基层的快速、简便的CSFV野毒鉴别检测方法. 相似文献
3.
两种荧光定量PCR方法检测猪瘟病毒的比较及应用 总被引：1，自引：0，他引：1
根据猪瘟病毒（Classical swine fever virus,CSFV）5端非编码区保守区域,设计猪瘟病毒特异性的引物和探针,建立了基于SYBR Green和TaqMan探针的两种荧光定量PCR检测方法。灵敏性和特异性试验结果表明,两种方法能够特异性地检测出不同型的猪瘟病毒,而对同属的牛病毒性腹泻病病毒以及其它一些主要猪病病原检测结果均为阴性。两种方法对猪瘟病毒C株的检测下限均为101TCID50,均高于常规的套式PCR。用携带该基因片段的重组质粒为模板进行检测,SYBR Green方法的检测下限为3×100copies,比TaqMan探针方法更加灵敏（3×101copies）。应用建立的SYBR Green方法对2009年采集于浙江地区不同猪场的20份病猪样品进行CSFV检测,有8份样品为阳性,与套式PCR方法检测结果一致。进一步应用SYBR Green方法对不同感染阶段细胞内病毒RNA复制水平进行检测,与病毒滴度的结果基本一致。因此,本研究建立的SYBRGreen荧光定量PCR检测方法能够应用于组织和培养细胞中猪瘟病毒的检测。 相似文献
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Hong Tian Xiangming Hou Jinyan Wu Yan Chen Youjun Shang Shuanghui Yin Keshan Zhang Xiangtao Liu 《Veterinary immunology and immunopathology》2014,157(1-2):59-64
Classical swine fever (CSF) is a highly contagious and often fatal disease of swine. It is caused by classical swine fever virus (CSFV), one of the members of the genus Pestivirus of the Flaviviridae family. The development of a safe and effective vaccine against the CSF is critical to pandemic control, this article shows a tandem-repeat multiple-epitope recombinant vaccine can protect pigs from CSFV challenge. That was composed as following: two copies each of glycoprotein E2 residues 693–707, 241–276 and 770–781, and two copies amino acid residues 1446–1460 of the non-structural protein NS2-3. In the challenge test, all of the swine vaccinated with Chinese vaccine strain (C-strain) were fully protected from a challenge with CSFV. However, after three successive vaccinations with the multiple-epitope recombinant vaccine, three out of five pigs were protected from challenge with CSFV (in terms of both clinical signs and viremia). These results demonstrate that multiple-epitope recombinant vaccine which carrying the major CSFV epitopes can induce a high level of epitope-specific antibodies and exhibit a protective capability that parallels induced by C-strain to a certain extent. 相似文献
5.
Sara Mu?oz-González Marta Perez-Simó Marta Mu?oz José Alejandro Bohorquez Rosa Rosell Artur Summerfield Mariano Domingo Nicolas Ruggli Llilianne Ganges 《Veterinary research》2015,46(1)
Classical swine fever (CSF) causes major losses in pig farming, with various degrees of disease severity. Efficient live attenuated vaccines against classical swine fever virus (CSFV) are used routinely in endemic countries. However, despite intensive vaccination programs in these areas for more than 20 years, CSF has not been eradicated. Molecular epidemiology studies in these regions suggests that the virus circulating in the field has evolved under the positive selection pressure exerted by the immune response to the vaccine, leading to new attenuated viral variants. Recent work by our group demonstrated that a high proportion of persistently infected piglets can be generated by early postnatal infection with low and moderately virulent CSFV strains. Here, we studied the immune response to a hog cholera lapinised virus vaccine (HCLV), C-strain, in six-week-old persistently infected pigs following post-natal infection. CSFV-negative pigs were vaccinated as controls. The humoral and interferon gamma responses as well as the CSFV RNA loads were monitored for 21 days post-vaccination. No vaccine viral RNA was detected in the serum samples and tonsils from CSFV postnatally persistently infected pigs for 21 days post-vaccination. Furthermore, no E2-specific antibody response or neutralising antibody titres were shown in CSFV persistently infected vaccinated animals. Likewise, no of IFN-gamma producing cell response against CSFV or PHA was observed. To our knowledge, this is the first report demonstrating the absence of a response to vaccination in CSFV persistently infected pigs. 相似文献
6.
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
7.
In this study, genomic sequences of pestiviruses available in GenBank were aligned to design three primer pairs and TaqMan probes: two targeting the NS5A region of the viral genome of classical swine fever virus (CSFV) for the differentiation of wild-type CSFV and hog cholera lapinized vaccine (HCLV) vaccine, and one targeting the 5'-untranslated region of bovine viral diarrhea virus 1 (BVDV-1). With these primers and probes, a triplex TaqMan real-time RT-PCR assay was developed for differentiating wild-type CSFV, the HCLV strain, and BVDV-1. The detection limit of the assay was 4.5 TCID(50) for wild-type CSFV, 10 TCID(50) for HCLV-strain CSFV, and 3.2 TCID(50) for BVDV-1. The triplex real-time RT-PCR had at least 98% (248 samples) agreement with other RT-PCR methods. The assay provides a sensitive tool for simultaneous detection and differentiation of wild-type CSFV and HCLV from BVDV-1. 相似文献
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MA Ping LI Da TANG De-yuan ZHANG Yuan-xin ZHANG Hua ZENG Zhi-yong LIU Xia WEI Guan-dong 《中国畜牧兽医》2016,43(9):2230-2239
For the distinguishment of wild-type and vaccine viruses of classical swine fever (CSF),a rapid and simple method was established,which laid the foundation for the purification of classical swine fever virus (CSFV) in large-scale farms.It was found that T-rich insertion sequence independently that 3'-NTR in Lapinized vaccine strain according to the genome sequence comparative analysis of CSF wild virus,vaccine virus and near origin virus published in GenBank,on the basis of this,two pairs of specific primers were designed in the upper and lower ends of the insertion sequence and duplex RT-PCR primers were designed.The anneal temperature of polymerese chain reaction (PCR) was optimized,then the two single and duplex RT-PCR method for the differentiation of wild-type and vaccine viruses of CSF were established.The sensitivity and specificity results showed that the minimum amounts of nucleic acid by the single RT-PCR were 2.2 pg (one pair of primers in single RT-PCR)and 1.7 pg (another pair of primers in single RT-PCR),respectively,and that of the duplex RT-PCR were 8.2 pg (wild-type virus of CSF) and 6.7 pg (vaccine virus of CSF), respectively, and no amplification of PRV,PRRSV,JEV,BVDV,PCV2 DNA/RNA were detected by these methods.Then the methods were used to detect 146 suspicious clinical samples,and the results showed that the positive rates of the mixed infection by wild-type and vaccine viruses of CSF in breeding sows,fattening pigs,nursery pigs and suckling piglets were 6.3%,7.4%,8.3%,8.6%, respectively.The results indicated that the single and duplex RT-PCR methods had high specificity,sensitivity,and good repeatability,and it had an important reference value for the purification of CSF in large-scale farms. 相似文献
9.
为建立一种对猪瘟病毒（classical swine fever virus,CSFV）临床样本快速、简便的检测技术,以区分猪瘟（classical swine fever,CSF）野毒和疫苗毒感染,为规模化猪场CSF净化奠定基础。通过对GenBank中CSF野毒、疫苗毒及近源病毒的基因组全序列比对,发现CSF兔化弱毒疫苗株3’-NTR独立存在富含T的插入序列,根据这一特点分别在该插入序列的上、下两端设计2对单一RT-PCR引物并选择特异保守区域设计了二重RT-PCR引物,优化筛选能够鉴别CSF野毒与兔化弱毒疫苗的PCR反应条件,建立了能鉴别CSF野毒和疫苗毒的单一与二重RT-PCR鉴别诊断方法。敏感性和特异性分析表明,单一RT-PCR检测CSFV各引物最低核酸检测量分别为2.2 pg（单一RT-PCR中的1对引物）和1.7 pg（单一RT-PCR中的另外1对引物）;二重RT-PCR检测CSFV各引物最低核酸检测量分别为8.2 pg（CSF野毒）和6.7 pg（CSF疫苗毒）,两种方法均检测不到PRV、PRRSV、JEV、BVDV、PCV2的DNA/RNA。采用该方法对146份可疑临床样品进行检测,结果表明CSF疫苗毒与野毒在能繁母猪、育肥猪、保育猪和哺乳仔猪中的二重感染率分别为6.3%、7.4%、8.3%、8.6%。本研究建立的单一与二重RT-PCR方法都具有敏感性强、特异性优、重复性好的特点,该研究对规模化猪场猪瘟的净化具有十分重要的参考价值。 相似文献
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针对CSFV基因组5＇端非编码区序列设计并合成了高度特异的一对引物和一条探针,用于猪瘟病毒实时荧光定量PCR检测方法的建立。将提取的病毒的总RNA做为模板进行反转录和PCR,将PCR产物克隆到pMDl8-T载体后进行大肠杆菌转化,提取阳性质粒做为标准品绘制标准曲线,成功地建立了特异性检测CSFV的荧光定量RT-PCR方... 相似文献
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本研究旨在建立猪瘟病毒野毒株和兔化弱毒疫苗株RT-PCR-RFLP鉴别检测方法。根据Shimen株设计1对特异性引物,建立猪瘟病毒RT-PCR-RFLP检测方法;对20份疑似猪瘟临床样品进行检测,并对检出的山东8株流行野毒株和2株疫苗株PCR产物进行克隆与序列分析,验证上述方法。结果RT-PCR扩增片段为825bp,产物经RFLP分析,野毒株的PCR产物能被ApaⅠ酶切为322bp和503bp 2个片段,兔化弱毒疫苗株则不能被酶切,检测出RNA的最低浓度为0.028 6μg.mL-1;8株流行野毒株都含GGGCCC序列(ApaⅠ酶切位点),2株疫苗株相应序列为GAGCCC,不能被ApaⅠ酶切;8株流行野毒株属于基因2群,2株疫苗株与HCLV遗传关系近,为基因1群。建立了可鉴别猪瘟病毒野毒株和兔化弱毒疫苗株RT-PCR-RFLP检测方法,为猪瘟的防控提供有效手段。 相似文献
13.
Patricia Renson Mireille Le Dimna André Keranflech Roland Cariolet Frank Koenen Marie-Frédérique Le Potier 《Veterinary research》2013,44(1):9
The conventional C-strain vaccine induces early protection against classical swine fever (CSF), but infected animals cannot be distinguished from vaccinated animals. The CP7_E2alf marker vaccine, a pestivirus chimera, could be a suitable substitute for C-strain vaccine to control CSF outbreaks. In this study, single oral applications of CP7_E2alf and C-strain vaccines were compared for their efficacy to induce protection against a CSF virus (CSFV) challenge with the moderately virulent Bas-Rhin isolate, in pigs as early as two days post-immunization. This work emphasizes the powerful potential of CP7_E2alf vaccine administered orally by a rapid onset of partial protection similar to that induced by the C-strain vaccine. Furthermore, our results revealed that both vaccinations attenuated the effects induced by CSFV on production of the pig major acute phase protein (PigMAP), IFN-α, IL-12, IL-10, and TGF-β1 cytokines. By this interference, several cytokines that may play a role in the pathogeny induced by moderately virulent CSFV strains were revealed. New hypotheses concerning the role of each of these cytokines in CSFV pathogeny are discussed. Our results also show that oral vaccination with either vaccine (CP7_E2alf or C-strain) enhanced CSFV–specific IgG2 production, compared to infection alone. Interestingly, despite the similar antibody profiles displayed by both vaccines post-challenge, the production of CSFV-specific IgG1 and neutralizing antibodies without challenge was lower with CP7_E2alf vaccination than with C-strain vaccination, suggesting a slight difference in the balance of adaptive immune responses between these vaccines. 相似文献
14.
ABSTRACT: Classical swine fever virus (CSFV) C-strain "Riems" escape variants generated under selective antibody pressure with monoclonal antibodies and a peptide-specific antiserum in cell culture were investigated. Candidates with up to three amino acid exchanges in the immunodominant and highly conserved linear TAV-epitope of the E2-glycoprotein, and additional mutations in the envelope proteins ERNS and E1, were characterized both in vitro and in vivo.It was further demonstrated, that intramuscular immunization of weaner pigs with variants selected after a series of passages elicited full protection against lethal CSFV challenge infection. These novel CSFV C-strain variants with exchanges in the TAV-epitope present potential marker vaccine candidates. The DIVA (differentiating infected from vaccinated animals) principle was tested for those variants using commercially available E2 antibody detection ELISA. Moreover, direct virus differentiation is possible using a real-time RT-PCR system specific for the new C-strain virus escape variants or using differential immunofluorescence staining. 相似文献
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Agüero M Fernández J Romero LJ Zamora MJ Sánchez C Belák S Arias M Sánchez-Vizcaíno JM 《Veterinary research》2004,35(5):551-563
The development and standardisation of a novel, highly sensitive and specific one-step hot start multiplex RT-PCR assay is presented for the simultaneous and differential diagnosis of African swine fever (ASF) and Classical swine fever (CSF). The method uses two primer sets, each one specific for the corresponding virus, amplifying DNA fragments different in length, allowing a gel-based differential detection of the PCR products. Universal detection of ASF and CSF virus strains was achieved through selection of primers in conserved viral genome regions. The detection range was confirmed by analysis of a large collection of isolates of the two viruses. The high specificity of the assay was proven by testing related viruses, uninfected cell line cultures and healthy pig tissues. Additional confirmatory tests of the ASF and CSF virus amplicon specificity, based on restriction endonuclease analysis with BsmA I or Ban II, respectively, are also described. The analysis of whole blood and serum samples from experimentally infected animals proved the usefulness of the method for an early diagnosis of both diseases, even before the appearance of the first clinical signs. A study of 150 positive field samples from several ASF and CSF outbreaks showed the suitability of this method for a rapid (less than five hours), sensitive and specific differential diagnosis in clinical samples. In addition, a highly sensitive and specific uniplex RT-PCR for CSFV was also developed and standardised as a powerful tool for fast and early diagnosis of the disease. 相似文献
18.
用荧光定量RT-PCR方法检测猪瘟病毒 总被引：4，自引：1，他引：4
为了建立能特异检测不同基因型猪瘟病毒(Classical swine fever virus,CSFV),同时又能区分其他瘟病毒的基因检测方法,本实验针对CSFV基因组5′端非编码区设计并合成了简并引物和TaqMan探针,在优化反应条件的基础上,成功地建立了特异检测CSFV的荧光定量RT-PCR检测方法。再以已知滴度的CSFV石门株血毒总RNA反转录产物建立标准品,该标准品可以用于定量临床样品中的CSFV滴度,所建立的荧光定量PCR方法可以灵敏地检测出10~(-0.82)个TCID_(50)病毒含量。最后用建立的方法对108份临床样品进行检测并同时进行病毒分离,荧光定量PCR方法检测出73份阳性样品且与病毒分离的符合率为100%,而常规RT-PCR只检测出54份阳性样品,表明本荧光定量RT-PCR法在检测猪瘟病料上具有潜在的应用价值。 相似文献
19.
A multiplex RT-PCR assay for the rapid and differential diagnosis of classical swine fever and other pestivirus infections 总被引：2，自引：0，他引：2
Heidy Díaz de Arce Lester J. Prez Maria T. Frías Rosa Rosell Joan Tarradas Jos I. Núez Llilianne Ganges 《Veterinary microbiology》2009,139(3-4):245-252
Classical swine fever is a highly contagious viral disease causing severe economic losses in pig production almost worldwide. All pestivirus species can infect pigs, therefore accurate and rapid pestivirus detection and differentiation is of great importance to assure control measures in swine farming. Here we describe the development and evaluation of a novel multiplex, highly sensitive and specific RT-PCR for the simultaneous detection and rapid differentiation between CSFV and other pestivirus infections in swine. The universal and differential detection was based on primers designed to amplify a fragment of the 5′ non-coding genome region for the detection of pestiviruses and a fragment of the NS5B gene for the detection of classical swine fever virus. The assay proved to be specific when different pestivirus strains from swine and ruminants were evaluated. The analytical sensitivity was estimated to be as little as 0.89 TCID50. The assay analysis of 30 tissue homogenate samples from naturally infected and non-CSF infected animals and 40 standard serum samples evaluated as part of two European Inter-laboratory Comparison Tests conducted by the European Community Reference Laboratory, Hanover, Germany proved that the multiplex RT-PCR method provides a rapid, highly sensitive, and cost-effective laboratory diagnosis for classical swine fever and other pestivirus infections in swine. 相似文献