猪传染性胃肠炎病毒TaqMan荧光定量RT-PCR检测方法的建立

根据猪传染性胃肠炎病毒和猪肌动蛋白的基因序列设计合成了引物和探针,通过对荧光定量RT-PCR反应条件的优化,建立了TaqMan荧光定量RT-PCR检测TGEV的方法。同时对37份现地病料进行检测并与常规RT-PCR方法、TGEV抗原快速检测试剂盒比较。结果,该方法的检测敏感性达到15.3拷贝/μL,且具有很好的特异性和重复性,而常规RT-PCR方法只能检测到1.53×103拷贝/μL。对37份现地病料的检测结果也表明该法(检出17份)比常规RT-PCR方法(检出12份)和TGEV抗原快速检测试剂盒(免疫层析法,检出10份)的敏感性高。     

用荧光定量RT-PCR方法检测猪瘟病毒

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。     

O型和Asia1型口蹄疫病毒感染RT-PCR鉴别诊断方法的建立

根据GenBank中O型和Asia1型口蹄疫病毒(Foot-and-mouth disease virus,FMDV)的vp3、vp1和2A基因序列,并与其它血清型FMDV的对应基因序列进行比较,设计用于扩增O型和Asia1型FMDV vp1基因的特异性引物,建立O型和Asia1型FMDV RT-PCR鉴别诊断方法。本方法首先用通用型引物进行RT-PCR,确定是否为FMDV感染,然后用特异性引物鉴别O型或Asia1型FMDV的感染。用vp1基因序列分析进行符合性试验,验证了该方法所具有的特异性和敏感性。本方法可用于O型和Asia1型FMD的快速诊断及流行病学调查。     

Assessment of real-time PCR as a method for determining the presence of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in different Solanaceae cultivars

Real-time PCR was used to detect and quantify Verticillium dahliae and to assess the susceptibility of four Capsicum annuum cultivars (Luesia, Padrón, SCM331 and PI201234) and the Capsicum chinense cv. C118 to this pathogen. The symptoms which developed after infection included stunting and yellowing, and were more acute in the cv. SCM331, which also suffered defoliation in later stages of the disease and in C118, which suffered severe stunting. Quantification of the pathogen DNA in roots 23 and 34 days post-inoculation (dpi) revealed that there were significantly higher amounts of Verticillium dahliae DNA in C118 than in the other cultivars, followed by SCM331, Padrón and PI201234. The lowest amounts of fungal DNA in roots were found in Luesia. In hypocotyls, the highest amounts of fungal DNA were found in SCM331, while Luesia, Padrón and PI201234 had much lower amounts, and C118 had intermediate levels. When a compatible versus an incompatible system was studied, using the near-isogenic tomato lines LA3030 (susceptible) and LA3038 (resistant to V. dahliae), we were able to detect fungal DNA in both lines. As expected, the fungus/plant DNA ratio was lower in LA3038 than in LA3030 and it decreased with time in LA3038. The amount of Verticillium dahliae DNA in the roots of LA3030 remained constant between days 23 and 34 post-inoculation, but increased 10-fold in collars. Finally, when real-time PCR was applied as a diagnostic method to samples from pepper plants, soil and water collected from farms in northwest Spain, we were able to detect V. dahliae DNA in these samples even when symptoms of the disease were not evident.     

二温式多重RT-PCR同删两种主要兰花病毒的研究

根据建兰花叶病毒(Cymbidium mosaic virus,Cy MV)和齿兰环斑病毒(Odontoglossum ringspot virus,ORSV)两种主要兰花病毒的保守序列设计特异性引物,建立了二温式多重RT-PCR同时检测两种病毒的检测方法。用该方法对感染两种病毒的植株总RNA模板进行扩增,结果同时得到2条大小与实验设计相符的769bp(Cy MV)、1000bp(ORSV)的特异性扩增带。用该方法对随机抽取的39个蝴蝶兰样品进行检测,结果11个样品检测出Cy MV,阳性率28.2%;24个样品检测出ORSV,阳性率61.5%;6个样品同时检测出两种病毒。     

侵染昆明玫瑰的李坏死环斑病毒的鉴定及其分子检测

采集昆明地区种植的花叶症状明显的玫瑰样品,运用现代分子生物学技术与常规技术相结合的方法,初步鉴定引起玫瑰花叶病的主要病毒病原为李坏死环斑病毒.该病毒引起玫瑰植株的系统花叶、畸形和皱缩等症状;电镜下病毒粒体为球形,直径为22～23nm;ELISA检测发现该病毒在植株芽、花粉和顶部叶片的浓度最高;同时,根据外壳蛋白的保守区利用Primer 5.0设计该病毒的特异引物,对该病毒进行分子检测,得到450bp的预期DNA片断,并在此基础上,进行了巢式RT-PCR的分子检测,表明巢式RT-PCR的检测能力最强.并通过序列的同源性分析得知该病毒的外壳蛋白与已知PNRSV的同源性为98.0%,进一步证明了该病毒为李坏死环斑病毒.     

猪繁殖与呼吸综合征病毒感染对猪肺泡巨噬细胞外源性抗原加工递呈相关分子和共刺激分子的影响

采用定量竞争PCR技术对猪繁殖与呼吸综合征病毒（PRRSV）感染后不同时相猪肺泡巨噬细胞（PAM）中外源性抗原加工递呈相关分子SLA-DR、Ii链和SLA-DM分子及共刺激分子CD40、CD80、CD86的mRNA转录动态进行了定量检测。结果表明,PRRSV感染后PAM的SLA-DR mRNA转录水平在3、7和14 d下调,低于对照组;感染后3 d,Ii链、SLA-DM和CD40的mRNA转录水平极显著下调（P〈0.01）;CD80和CD86mRNA转录水平也在感染后3 d明显下调（P〈0.05）。用流式细胞术检测PAM表面的SLA-DR抗原的结果表明,在感染后3 d短暂上升,7 d之后一直低于对照组。由此说明,PRRSV感染早期对猪肺泡巨噬细胞的外源性抗原加工和递呈功能产生显著影响,导致其外源性抗原递呈功能下降,进而影响机体的体液免疫应答。     

湿段天气在小麦赤霉病定量预报中的应用

通过本县小麦感病期内逐日气象观测资料统计分析,发现在该期内各日２：００、８：００、２０：００的空气相对湿度达８５％,１４：００达８２％,且在此湿度标准上持续３０ｈ以上,平均气温达１２℃的湿段天气累计持续时间长,小麦赤霉病发生重。依据湿段天气段在小麦感病的穗期开展赤霉病的短期定量预测,１０年次的回报病穗率误差为２个百分点左右,７年次的预报病穗率误差为０．２９￣５．２个百分点。     

Use of Degenerate Primers in a RT-PCR Assay for the Identification and Analysis of Some Filamentous Viruses, with Special Reference to Clostero- and Vitiviruses of the Grapevine

RT-PCR with degenerate primers was used for the screening of the genome of some members of the Closterovirus, Vitivirus and Trichovirus genera. Two sets of primers, targeted to conserved sequences of the heat shock protein 70 homologue of closteroviruses or to the RNA dependent RNA polymerase genes of tricho- and vitiviruses, amplified the expected fragments from total RNA extracts or double-stranded RNAs of infected plants. Amplified cDNAs were cloned, sequenced and phylogenetically analyzed. Results support the allocation of grapevine viruses A, B, D and heracleum latent virus (HLV) in the genus Vitivirus, whereas, the detection of a HSP70 homologue in grapevine leafroll-associated viruses agrees with their assignment in the genus Closterovirus. The use of degenerate primers for the identification of grapevine viruses belonging to Vitivirus and Closterovirus genera is envisaged.     

四君子汤对仔猪胸腺及脾脏生长激素受体mRNA表达的影响

选用90头新生"杜×长×大"三元杂交仔猪,随机分为3组,每组30头,分别为中药I组(中药添加剂量10 g/kg),中药Ⅱ组(中药添加剂量20 g/kg),对照组(基础日粮组),试验期为31 d.45日龄时每窝随机选取6头仔猪屠宰后,分别采集胸腺和脾脏.采用反转录聚合酶链式反应(RT-PCR)方法,定量分析仔猪胸腺及脾脏中生长激素受体(GHR)mRNA的相对丰度.结果显示,与对照组相比较,2种剂量中药组的仔猪胸腺及脾脏中生长激素受体mRNA的含量均不同程度地提高,中药Ⅱ组效果更为显著.