PCR技术在猪圆环病毒2型检测中的应用

参照Gen Bank中已发表的猪圆环病毒2型(PCV-2)ORF2基因序列,设计合成1对引物,建立了检测猪场疑似病例临床样本中PCV-2的PCR方法。结果显示,PCV-2的ORF2扩增产物经琼脂糖凝胶电泳,呈现1条630 bp的特异性条带,与预期目的片段大小一致。应用该方法对安徽省3个猪场的30份病料进行了PCV-2检测,结果有9份检测出PCV-2,阳性率为30%。结果表明,该试验建立的PCR方法,是一种快速、灵敏、高效的PCV-2检测技术。     

屠宰生猪PCV-2感染的检测与基因型分析

为了解屠宰生猪中猪圆环病毒2型(PCV-2)的带毒状况及PCV-2的主要基因型,在分析PCV-2分离株基因序列的基础上分别设计了检测PCV-2、PCV2-A和PCV2-B的引物,建立了各自的PCR检测方法。结果表明,建立的PCV-2、PCV2-A、PCV2-B的PCR检测方法,特异性较好,可用于临床病料的检测;通过对临床采集的49分生猪血样的检测发现,PCV-2的阳性率为24.49%,均属于PCV2-B。检测结果表明,当地检测猪群中感染的PCV-2主要为PCV2-B毒株,在该病的防控方面应引起注意。     

猪圆环病毒2型和3型双重PCR鉴别检测方法的建立及应用

为建立一种鉴别检测猪圆环病毒2型(PCV-3)和3型(PCV-3)的方法,本研究根据GenBank中PCV-2、PCV-3基因序列分别设计合成特异性检测引物,经过双重PCR反应条件的优化,建立了鉴别检测PCV-2和PCV-3的双重PCR方法。该方法对PCV-2、PCV-3、PCV-2/PCV-3混合物可分别扩增出300 bp、518 bp、300 bp和518 bp的特异性条带,检测PRV、PPV、PRRSV、CSFV、TGEV、PEDV均为阴性,对PCV-2/PCV-3混合物的检测灵敏度达2.1 ng DNA,与PCV-2和PCV-3单项PCR方法的符合率均为100%。该方法在检测136份临床病料样品的应用中,PCV-2阳性率达23.53%(32/136),PCV-3阳性率达18.38%(25/136),PCV-2/PCV-3混合感染率达7.35%(10/136),说明青海省部分地区猪群中存在PCV-2、PCV-3的流行,且存在PCV-2和PCV-3的混合感染。表明该双重PCR具有良好的特异性、敏感性、重复性、准确性和临床适用性,可以用于临床病料中PCV-2和PCV-3的快速低含量鉴别检测,将为PCV-2和PCV-3感染的临床鉴别诊断和流行病学监测提供了一种简便适用的方法,为我国猪圆环病毒病的综合防控提供技术支持。     

猪伪狂犬病病毒和猪圆环病毒2型多重PCR检测方法的建立与初步应用

为了建立可同时检测猪伪狂犬病病毒(PRV)和猪圆环病毒2型(PCV-2)的多重PCR检测方法,本研究根据GenBank中登录的PRV gE基因和PCV-2 ORF2基因的核苷酸序列,分别设计了2对特异性引物PRV-S/PRV-A和PCV-2-S/PCV-2-A,产物分别为270 bp和189 bp。检测结果显示,该方法具有较高特异性,检测PRV和PCV-2的DNA最低量均为1×10~(-4)μg/μL。对136份临床上疑似为PRV和PCV-2感染病料样品进行检测的结果表明,本研究建立的多重PCR检测方法可用于临床上PRV和PCV-2引起的单纯或混合感染的传染病病原学诊断。     

猪传染性胸膜肺炎放线杆菌和猪圆环病毒2型二重实时荧光定量PCR检测方法的建立

以胸膜肺炎放线杆菌(App)apx ⅣA基因和猪圆环病毒2型(PCV-2)ORF2基因为靶基因,分别设计特异性引物和探针,建立App和PCV-2的二重荧光PCR方法。App引物扩增的目的片段大小为132bp,PCV-2为169bp。建立的方法可在同一反应体系中同时对App和PCV-2进行定量检测鉴别,其敏感性分别为1×101拷贝和1×100拷贝,对其他猪病病原核酸的检测为阴性。利用该方法对10份人工感染临床样品进行检测试验,其结果符合率达100%,说明该方法可用于对临床样品中App和PCV-2的检测。     

猪圆环病毒1型和2型a与b基因型三重PCR鉴别方法的建立及应用

根据PCV-1、PCV-2a和PCV-2b差异序列,设计了3对特异性引物,通过优化反应条件,建立了可以用于PCV检测及PCV-1、PCV-2a和PCV-2b分型的PCR方法。结果表明,三重PCR检测的最低敏感度可达4.87×107 copies/μL(PCV-1)、2.83×107 copies/μL(PCV-2a)和2.96×106 copies/μL(PCV-2b)。引物特异性良好,各引物之间没有交叉反应,对伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)和猪瘟病毒(CSFV)检测均为阴性。应用该方法对采自陕西省的56份样品进行检测,PCV-1检出率57.14%(32/56),PCV-2a检出率1.78%(1/56),PCV-2b检出率42.86%(24/56),多重PCR检测结果与单PCR检测结果的符合率达99%以上,可用于猪圆环病毒的临床检测及流行病学监测等。     

猪圆环病毒2型PCR检测方法的建立及其在临床检测中的应用

选择猪圆环病毒2型(PCV-2)基因保守区设计1对引物P1和P2,扩增536 bp的片段,该方法可以特异地检测出PCV-2的DNA,而对猪细小病毒、猪瘟病毒、猪伪狂犬病毒及未接PCV-2的PK-15细胞均呈阴性;该法能检测出29 ng/L的病毒DNA。对扩增片段进行测序,结果表明扩增片段属于PCV-2。应用该方法对2008年度上海及周边地区送检的91份临床样本进行了PCV-2的检测,结果表明,阳性样本为52份,阳性率为57.14%。研究结果表明,建立的PCR方法检测PCV-2具有较好的敏感性和特异性,可用于PCV-2感染疑似病例的诊断及其分子流行病学调查。     

PCR技术在猪圆环病毒2型检测中的应用

根据猪圆环病毒2型（PCV-2）ORF2基因序列,设计并合成了一对引物,以本室分离鉴定的PCV-2为模板,筛选最佳反应条件,建立了检测PCV-2的PCR方法。应用该方法对PCV-2进行基因扩增,获得长度为559bp的特异性目的DNA片段,而对PRRSV、CSFV、PPV等病毒进行同条件检测,结果均为阴性,无交叉反应;敏感性试验表明,该体系可检测到10．8pg的PCV-2基因组DNA。对2004年-2005年期间送检的81份临床样品进行检测,阳性率为22．2％。表明所建立的PCR技术可用于PCV感染的诊断和流行病学调查。     

猪细小病毒、伪狂犬病病毒和猪圆环病毒2型多重PCR检测方法的建立及应用

根据猪细小病毒(PPV)、伪狂犬病病毒(PRV)和猪圆环病毒2型(PCV-2)的基因序列,分别选取各自的保守区段设计引物,通过反应条件的优化,建立了检测PPV、PRV和PCV-2的多重PCR方法.用建立的方法对采自陕西省部分猪场的286份病料及血样进行检测,从对临床健康猪全血样品中PPV、PRV和PCV-2的检测结果看...     

猪圆环病毒2型PCR检测方法的建立及其应用

参照国内外已发表的猪圆环病毒2型（PCV-2）的ORF1的基因序列及其相关的PCR检测方法设计一对引物,扩增目的片段为493bp。通过对PCR检测方法的特异性、敏感性、重复性试验,建立了一种PCV-2的PCR检测方法。应用此方法对临床样品进行了检测,阳性检出率达51.18%。该方法具有快速、敏感、特异等优点,可用于PCV-2的检测和流行病学调查等。     

Porcine circovirus-2 DNA concentration distinguishes wasting from nonwasting pigs and is correlated with lesion distribution, severity, and nucleocapsid staining intensity.

The emergence of severe porcine circoviral disease in North America is associated with Porcine circovirus-2 genotype b (PCV-2b), which has led to speculation that PCV-2b is more virulent than PCV-2a. The objectives of this study were to 1) correlate the PCV-2 DNA concentration and lesions in wasting (WST) and age-matched healthy (HLTH) pigs from 2 clinically affected farms, and unaffected (UNFCT) pigs from a farm with no prior clinical or diagnostic history of PCVD; and 2) to determine the initial estimates of sensitivity and specificity of PCV-2 quantitative polymerase chain reaction (qPCR). PCV-2b was confirmed in all 3 farms. Compared with HLTH pigs, WST pigs demonstrated significantly more prevalent thymic atrophy, failure of normal pulmonary collapse, and ascites (P < 0.017 for all). The HLTH and UNFCT pigs had significantly more pronounced lymphoid germinal centers and proliferative paracortical T-dependent zones, compared with WST pigs (P < 0.017). Across all tissues, PCV-2 DNA concentrations were significantly higher in WST compared with HLTH and UNFCT pigs (P < 0.017 for all). The PCV-2 DNA concentrations were strongly correlated with PCV-2 nucleocapsid staining intensity in lymph node, spleen, Peyer's patches, lung, liver, and kidney (0.60 < or = r < or = 0.84). In the current study, the PCV-2 DNA log10 cutoff concentrations best able to distinguish WST from HLTH and UNFCT pigs were between 7.0 and 8.0 per gram for tissues, and between 4.0 and 5.0 per milliliter for sera. The presence of PCV-2b in UNFCT pigs is evidence that PCV-2b by itself is not sufficient to induce severe disease.     

Clinical and pathological observations on pigs with postweaning multisystemic wasting syndrome

The aim of this work was to characterise the lesions and agents present in clinically normal and clinically affected pigs on a farm during an outbreak of postweaning multisystemic wasting syndrome (PMWS), and to evaluate the diagnostic techniques for detecting porcine circovirus type 2 (PCV-2) and other microorganisms. Four pigs in the early stage and 11 pigs in the late stage of the disease, and eight clinically normal pigs were necropsied. Samples of lymphoid tissue and serum were also obtained from 12 slaughter pigs from the same farm. The tissues were examined histopathologically, and in situ hybridisation, serology and PCR were used to detect porcine circovirus type 1 (PCV-1) and/or PCV-2 in tissues and/or sera. The presence of porcine reproductive and respiratory syndrome virus (PRRSV), Aujeszky's disease virus (ADV) and porcine parvovirus (PPV) were also investigated. Characteristic microscopical lesions of PMWS were observed in the lymphoid tissues of the pigs in all three necropsied groups; the lesions were most common and severe in the pigs in the early stage of the disease, less so in the pigs in the late stage of the disease, and least in the clinically normal pigs. PCV-2 infection was detected in all the necropsied pigs by in situ hybridisation and PCR. Only three pigs had the PCV-1 genome in serum or lymph node tissue. In contrast, the slaughter pigs had no microscopical lesions and no PCV-2 nucleic acid in their serum or tissues, and only one of them had the pCV-1 genome in its serum. Immunohistochemical, serological and PCR studies revealed that PRRSV and ADV were also present on the farm during the outbreak.     

猪圆环病毒2型疫苗研究进展

猪圆环病毒2型(PCV-2)感染是一种新发现的猪传染病,以5周龄～12周龄仔猪高发病率、高死亡率及导致机体淋巴系统损伤与免疫缺陷为主要特征。PCV-2感染在世界各养猪国家广泛流行,给各国养猪业造成了巨大的经济损失。PCV-2疫苗研究已成为国内外学者的研究焦点。迄今为止,西欧与美国已有PCV-2疫苗注册上市,主要为灭活疫苗与亚单位疫苗。由于灭活疫苗与亚单位疫苗成本高,且注射次数多,容易造成机体应激,因此,弱毒疫苗、活载体疫苗及核酸疫苗等新型疫苗是目前研究的重点,并取得了长足的进展。论文主要概述了PCV-2疫苗研究的最新进展,以期为PCV-2疫苗研究提供一定的参考资料。     

华南地区猪圆环病毒和猪繁殖与呼吸综合征病毒检测分析

为了解华南地区猪圆环病毒及猪繁殖与呼吸综合征病毒的最新流行情况,采集了华南地区11个规模猪场各饲养阶段猪血清807份,用套式PCR(nPCR)检测猪圆环病毒1型(PCV-1)和猪圆环病毒2型(PCV-2);采集了若干猪场2010年1月至2011年8月期间有咳嗽、喘气、消瘦及疑似PDNS等临床症状的猪血清312份,以及无临床症状猪血清104份,以nPCR检测PCV-2,以一步法反转录PCR(RT-PCR)检测猪繁殖与呼吸综合征病毒。结果发现,所调查的11个规模猪场中只有5个检出PCV-1,所有猪场均检出PCV-2。PCV-2阳性率为30.61%,而PCV-1阳性率仅为4.21%;经产母猪和7周龄以上保育猪PCV阳性率最高;有临床症状的猪血清PCV-2阳性率为58.65%,PRRSV阳性率为37.82%,PCV-2阳性猪群中有39.89%的猪同时感染PRRSV;有症状猪群7月～9月的PCV-2感染率最高,而1月～3月最低;无临床症状猪血清PCV-2阳性率为27.9%,PRRSV阳性率为0.96%,PCV-2与PRRSV无混合感染。证明PCV尤其是PCV-2在华南地区仍广泛传播并流行,而且PCV-2与PRRSV混合感染致病情况较多。PCV-2的感染率与季节有一定的相关性,种猪带毒情况严重。     

猪瘟疫苗在猪圆环病毒2型阳性猪场的免疫效果观察

为了研究猪圆环病毒2型(PCV-2)感染对猪瘟疫苗免疫效力的影响,对PCR证实为PCV-2阳性的试验猪进行猪瘟疫苗的免疫,分别在免疫后第1、3、7、14、21、28和63天对试验猪进行猪瘟病毒(CSFV)和PCV-2的抗体检测。检测结果表明PCV-2阳性猪在猪瘟疫苗免疫后均未能产生有效的CSFV抗体,从免疫猪的血液和内脏组织中也未能检测到CSFV核酸。虽然只能从1头PCV-2阳性猪的血清中检测到PCV-2抗体,但是却能从全部实验猪的淋巴结中检测到PCV-2的核酸。试验猪的病理组织学观察和白细胞计数也表明PCV-2阳性猪的淋巴结呈典型的PCV-2感染的病理变化,且白细胞数量显著低于健康猪。表明PCV-2的感染会对猪的免疫系统造成损害从而抑制猪瘟疫苗的免疫效果。     

Porcine circovirus-2 and concurrent infections in the field

Porcine circovirus-2 (PCV-2) is the necessary cause of post-weaning multisystemic wasting syndrome (PMWS) in swine; however, a variety of co-factors, including other infectious agents, are thought to be necessary in the full expression of disease. Porcine parvovirus (PPV) was found in the inoculum used in the first experiments to reproduce PMWS in gnotobiotic swine. Retrospective and prospective studies in the field and laboratory have demonstrated PCV-2 can act synergistically with PPV to enhance the severity of PMWS. PCV-2 has been shown to play a role in the porcine infectious disease complex (PRDC). Other co-infecting agents with PCV-2 in the lung include, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV) and Mycoplasma hyopneumoniae. Exposure of pregnant sows to PPV, PRRSV, or encephalomyocarditis virus may interact with PCV-2 infected foetuses. The severity of hepatic lesions in PCV-2 infected pigs may be enhanced by co-infection with agents such as swine hepatitis E virus and Aujezsky's disease virus. Additional studies are required to determine the mechanistic basis for the interaction of PCV-2 with other agents in the pathogenesis of the various clinical syndromes that have been associated with PCV-2 infection.     

Quantitative polymerase chain reaction for Porcine circovirus-2 in swine feces in a Porcine circovirus disease-affected commercial herd and a nonaffected commercial herd

This study examined if pigs in a Porcine circovirus disease (PCVD)-affected herd (n = 100) had shed more Porcine circovirus-2 (PCV-2) in their feces than pigs in a PCVD-nonaffected herd (n = 101), and if differences in shedding among production stages within and between the herds existed. The PCV-2 shedding was quantified by real-time polymerase chain reaction. The highest median PCV-2 shedding was found in the nursery of the PCVD-affected herd and in the grower of the PCVD-nonaffected herd. The PCV-2 shedding was significantly higher in earlier stages (newly weaned, nursery, and pregrower) in the PCVD-affected herd (Wilcoxon rank sum; P < 0.001) compared with the PCVD-nonaffected herd. Porcine circovirus-2 DNA was not detected in a significant proportion of lactating sows (parity ≥ 3) in the PCVD-nonaffected herd (Fisher’s exact test; P = 0.001). The results of this study suggest there may be an association between the presence of PCV-2 in the feces of lactating sows and increased PCV-2 shedding in younger pigs.     

Retrospective study of porcine circovirus 2 infection in Japan: seven cases in 1989

Retrospectively, we demonstrated that seven pigs necropsied in 1989 had characteristic porcine circovirus 2 (PCV-2) lymphoid tissue lesions of severe lymphoid depletion, syncytial giant cell formation, and intracytoplasmic inclusion bodies in macrophages. Immunohistochemically, PCV-2 antigen was detected in these tissues and the distribution of positive staining corresponded to the distribution of inclusion bodies. Electron microscopy demonstrated viral particles compatible with porcine PCV-2. Therefore, the disease occurred in Japan as early as 1989.     

The effect of vaccination against Porcine reproductive and respiratory syndrome virus (PRRSV) on the Porcine circovirus-2 (PCV-2) load in porcine circovirus associated disease (PCVAD) affected pigs

A diagnostic project was initiated across the United States in 2006 to improve the understanding of porcine circovirus associated diseases (PCVAD) as well as to identify co-factors in PCVAD-affected farms. A Porcine circovirus-2 (PCV-2) DNA real-time polymerase chain reaction quantitation (qPCR) was established according to a published method and sera from a total of 23pig farms across the United States were examined for viral loads for PCV-2 and analyzed for any possible effects of Porcine reproductive and respiratory syndrome virus (PRRSV) vaccination on this parameter. Vaccination against PRRS resulted in significantly lower viral loads for PCV-2 in animals 13 wk or older compared with nonvaccinated animals, but vaccination of pigs against PRRS had no effect on qPCR results for PCV-2 in 4- to 12-week-old pigs. Interestingly, PRRS vaccinates had significantly lower viral loads when peak wasting disease was observed in the herds. The qPCR method for PCV-2 proved to be an important tool for help in the antemortem diagnosis of PCVAD as well as in the monitoring of infection dynamics.