Establishment and Application of a Multiplex RT-PCR Assay for Detection of PEDV,TGEV and PRoV

In this study,a multiplex RT-PCR assay was established to differentially detect porcine epidemic diarrhea virus (PEDV),porcine transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PRoV) after optimization of the reaction conditions.Three pairs of primers PEDV-N,TGEV-M and PRoV-VP6 were designed for specifically amplifying PEDV N gene,TGEV M gene and PRoV VP6 gene,respectively.The assay could specifically amplify PEDV,TGEV and PRoV,but not classical swine fever virus (CSFV),porcine foot and mouth disease virus (FMDV),pseudorabies virus (PRV),porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2).The detection limits of PEDV,TGEV and PRoV standard recombinant plasmids were 1.41×10³,1.41×10² and 1.41×10³ copies/μL,respectively.The repeated reaction under the same conditions obtained uniform results.The assay was used to detect a total number of 190 clinical samples,of which 42 (22.11%) samples were positive for PEDV,58 (30.53%) samples for TGEV and 34 (17.89%) samples for PRoV,and there were mixed infection among these viruses.The results indicated that this multiplex RT-PCR assay had the advantages of sensitivity,specificity and repeatability and provided a useful tool for differential detection and epidemiological investigation of PEDV,TGEV and PRoV.     

Establishment of a Duplex Real-time RT-PCR Assay for the Differential Detection of Nipha Virus and Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

To establish a rapid,sensitive and specific assay for the differential detection of Nipah virus (NiV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV),a duplex Real-time RT-PCR was developed with specific primers and probes targeting to the special sequences of NiV M gene and HP-PRRSV nsp2 gene by optimization of reaction conditions.The performance of the assay was linear ranging from 4.6×10¹ to 4.6×10⁷ copies/μL for RNA standard control of NiV M (NiV-M-RNA) and from 4.1×10¹ to 4.1×10⁸ copies/μL for RNA standard control of HP-PRRSV nsp2 (HP-PRRSV-nsp2-RNA),and detection limits of the assay was 46 copies for the NiV-M-RNA and 4.1 copies for the HP-PRRSV-nsp2-RNA,respectively.The coefficients of variation (CVs) of both inter-assay and intra-assay repeatability were less than 2.0%,showing good repeatability.The assay was able to specifically detect NiV and HP-PRRSV simultaneously without cross-reaction with classical swine fever virus (CSFV),porcine epidemic diarrhea virus (PEDV),swine influenza virus (SIV),porcine parvovirus (PPV),pseudorabies virus (PRV) and porcine circovirus type 2 (PCV2).Of the 236 samples from pigs for both NiV and HP-PRRSV detection by the established assay,all the samples were negative for NiV,8 samples were HP-PRRSV positive.In conclusion,this assay offers a useful approach for the differential detection of NiV and HP-PRRSV in clinical specimens from the pigs.     

神经肽S及其受体在猪免疫器官中的表达及分布

本研究采用半定量RT-PCR方法,对神经肽S(NPS)及其受体(NPSR)mRNA在猪免疫器官中的表达水平进行检测。结果表明,NPSmRNA在胸腺、脾、空肠淋巴结和软腭扁桃体中均有表达,且在脾和胸腺中的表达水平较高;NPSR mRNA在脾脏的表达水平最高。进一步利用免疫组织化学法对NPS在猪免疫器官中的分布定位进行了研究,在猪脾脏和空肠淋巴结均有NPS免疫阳性细胞分布,但在软腭扁桃体和胸腺中未见NPS免疫阳性细胞。再进行免疫接种猪传染性胃肠炎疫苗,运用半定量RT-PCR技术检测免疫接种后不同时间(3、7、14 d)NPS及其受体mRNA在猪免疫器官中表达水平的变化规律。结果表明,NPS mRNA和NPSR mRNA在胸腺、脾、淋巴结和扁桃体中均有表达,且在一定时间范围内呈现先升高后下降的变化规律,但对照组无明显的升高和下降现象。上述结果提示,猪NPS可能参与免疫功能的调节。     

小型猪复合麻醉剂及其特异性颉颃剂交互作用对大鼠不同脑区iNOSmRNA转录的影响

为研究小型猪专用复合麻醉剂（XFM）及其特异性颉颃剂交互应用对大鼠不同脑区iNOS mRNA转录的影响,将48只SD大鼠随机分为生理盐水对照组（C）和XFM与颉颃剂交互作用组（MJ）,MJ组分为早期交互（MJ1、MJ2）和晚期交互（MJ3、MJ4）2个亚组。各组大鼠到达预定时间点后分别采取脑组织,应用实时荧光定量PCR技术检测各组织中iNOS mRNA表达量。结果显示,XFM及其特异性颉颃剂交互应用时,大脑、小脑、海马、脑干和丘脑中iNOS mRNA转录均受到显著抑制（P〈0.01）,虽能逐渐回升,但在试验选取时间点内只有小脑与海马中iNOS mRNA转录恢复正常。结果表明,XFM及其特异性颉颃剂在交互作用时能够显著抑制中枢神经系统中iNOS mRNA的转录,部分脑区可以完全恢复,这可能与XFM及其特异性颉颃剂交互作用机制有关。     

Distribution and properties of geographically distinct isolates of sugar beet yellowing viruses

From a total of 261 yellow sugarbeet leaves collected from 10 countries representing three continents, the incidence and distribution of strains of Beet mild yellowing virus (BMYV), Beet chlorosis virus (BChV) and Beet yellows virus (BYV) were analysed using serological and molecular methods. BMYV was found in all countries except Greece, and more frequently in the northern and western areas of Europe, whereas BYV predominated in Turkey, Spain, Greece, the USA and Chile. BChV, originally found in the USA and the UK in 1989, was identified in France, Spain, the Netherlands and Chile. Nine sugar beet poleroviruses, plus a reference isolate of Turnip yellows virus (TuYV, syn. Beet western yellows virus ), were further characterized and compared. Isolates obtained from sugar beet infected this species, but not oilseed rape or lettuce; all isolates except one infected Capsella bursa-pastoris . The coat-protein sequences of these isolates were highly similar, with the consensus sequence representing 89% of nucleotide residues. Within the coat-protein gene, two regions were identified that could represent specific epitopes to which monoclonal antibody BYDV-PAV-IL-1 could bind; this antibody is used to distinguish beet poleroviruses in ELISA. Comparison of the sequences at the 5' end showed that sequence homology existed only between isolates with the same host range. The first sequence data of polerovirus isolates from Chile are presented, showing that the coat protein and the 5' end of their genomes are highly similar to those of BMYV isolates found in Europe. Chilean polerovirus isolates may have been imported from the northern hemisphere in sugar beet breeding material.     

苹果茎沟病毒部分分离物的生物学特性与分子鉴定

通过昆诺藜接种鉴定和ELISA检测,从梨和苹果上分离获得苹果茎沟病毒(Apple stem grooving virus,ASGV)23个分离物.采用TC-RT-PCR对这些分离物进行扩增,均获得特异的扩增片段,PCR产物经5%PAGE电泳,出现大小约500、530和600bp的3种迁移率不同的泳动带型.根据PCR产物电泳迁移率的差异,选取3个来源于梨的分离物P-L4、P-6-1-17和P-3-2-67的PCR产物进行克隆与序列测定.经BLAST搜索,3个分离物的扩增片段与苹果分离物P-209的CP基因3′端核苷酸序列同源性分别为92.2%、90.4%和88.4%.3个分离物间的核苷酸序列也有较大差异,P-L4/P-6-1-17为95.5%、P-L4/P-3-2-67为90.4%、P-6-1-17/P-3-2-67为88.6%.     

桃潜隐花叶类病毒RT-PCR和分子杂交检测技术的建立

本研究从武汉周边采集表现典型花叶症状的桃样品,提取总RNA为模板,采用RT-PCR方法对这些样品进了分析,结果显示所分析的5个样品(P1～P5)均获得了预期大小约为337bp的目标扩增条带,表明样品均带有PLMVd.通过回收RT-PCR产物,用生物素标记制备探针,分别采用DNA斑点杂交、RNA斑点杂交和组织印迹杂交3种杂交方法对这些样品进行检测比较,3种杂交方法中,组织印迹杂交操作步骤相对简单快捷,适合于对PLMVd进行大田快速检测.     

RT-PCR结合限制性内切酶分析法区分NDV强弱毒株

通过设计的特异引物，采用RT-PCR对1996年以来分离的45株国内新城疫病毒分离株进行鉴定和分析，并与其MDT实验结果进行比较，建立了RT-PCR结合BglⅠ内切酶分析区分新城疫强弱毒的方法。此方法操作简便、迅速，不仅能区分新城疫的强弱毒株，还可诊断AMPV-1引起的其他禽类的感染。