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。     

<Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis>: a new potential biocontrol agent of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis>, causal agent of potato brown rot

Stenotrophomonas maltophilia was isolated from the rhizosphere of eggplant in the Nile Delta of Egypt, and its antagonistic potential against Ralstonia solanacearum race 3 biovar 2, the causal agent of potato brown rot, was in vitro evaluated on KB agar medium and in vivo on potato plants. In vitro, four isolates of S. maltophilia (PD3531, PD3532, PD3533, and PD3534) appeared antagonistic. The isolate (PD3533) was screened as the most promising antagonist for the in vivo tests. In the greenhouse, the antagonist was applied directly to soil or by bacterization of potato eyepieces. Stenotrophomonas maltophilia significantly suppressed potato brown rot in Egyptian clay soil but not in Dutch clay soil. Survival of a rifampicin and chloramphenicol-resistant S. maltophilia strain PD4560 was investigated in two pairs of clay soils, conventionally and organically managed, from Egypt and the Netherlands. The survival of S. maltophilia was significantly less in Dutch than in Egyptian soils, while the converse occurred for R. solanacearum. These results are in agreement with those obtained in the in vivo biocontrol tests. In conclusion, S. maltophilia may be useful for control of brown rot in the area where it was originally isolated, the Nile Delta in Egypt.     

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.     

马泰勒虫不同PCR检测方法的比较

为寻求一种快速、有效的马泰勒虫PCR检测方法，用Bec-UF2、Equi-R；EMA-1F、EMA-1R两对引物对56份马血液样本中马泰勒虫的核蛋白体基因和表面蛋白基因进行了常规PCR方法检测，用EMA-5、EMA-6；EMA-7、EMA-8两对引物对56份马血液样本中马泰勒虫的表面蛋白基因进行了PCR和套式PCR方法检测。结果显示，以Bec-UF2、Equi-R为引物的PCR方法的阳性检出率为57．1％（32／56），以EMA-5、EMA-6；EMA-7、EMA-8为引物的套式PCR方法的阳性检出率为51．8％（29／56），以EMA-1F、EMA-1R为引物的PCR方法的阳性检出率为17．9％（10／56）。结果表明，以Bec-UF2、Equi-R为引物的常规PCR方法为检测马泰勒虫的最佳方法。     

狂犬病毒RT-PCR检测方法的建立

根据狂犬病毒保守序列设计引物,利用反转录聚合酶链式反应(RT-PCR)建立了一种检测狂犬病毒的方法,以期实现对严重危及人和动物生命安全的狂犬病毒直接检测。     

羊草乙醛脱氢酶（ALDH）基因片段的克隆及表达分析

[研究目的]克隆羊草的乙醛脱氢酶ALDH基因片段,研究该基因在不同条件下的表达情况。[方法]采用RT-PCR技术克隆羊草的ALDH基因片段,并对其核苷酸和氨基酸序列进行分析,采用RealTimeRT-PCR方法研究该基因的表达。[结果]获得了羊草的ALDH基因片段,长度为675bp,编码225aa。核苷酸序列比较表明,与水稻ALDH1a序列(AB037421)同源性为86%,与玉米RF2C(AF348413)同源性为85%。BLASTp分析,该序列与水稻、玉米、拟南芥的乙醛脱氢酶一致性分别高达87%、86%、60%,含有醛脱氢酶基因家族的保守结构域。RealTimeRT-PCR数据表明,在诱导条件下该基因的表达量呈先升高后降低的趋势。总体上来说,该基因对盐的响应要高于干旱和冷冻。[结论]成功获得了羊草ALDH基因片段,并研究了该基因的表达情况,为进一步克隆羊草ALDH全长基因奠定了基础。     

不同日龄仔猪骨髓组织抗菌肽Protegrin-1mRNA的表达

分别于出生当天、3、14、23和28日龄随机屠宰杜×长×大三元杂交公猪4头，采集股骨骨髓组织。用一优化的RT—PCR方法，以18S rRNA作内标，定量分析不同日龄仔猪骨髓组织抗菌肽Protegrin-1（PG-1）mRNA表达的差异。结果表明，不同日龄仔猪骨髓组织PG-1mRNA相对丰度有明显的差异。出生时最低，随着日龄的增加，其表达显著增加。     

猪TGFβ1基因及其受体组织表达特性的研究

以妊娠母猪为研究材料,采用RT-PCR半定量方法对猪转化生长因子β1(TGFβ1)及其3种受体组织表达特点进行研究。结果表明:TGFβ1在11种组织中的表达量,由高到低依次为肾上腺、下丘脑、肌肉、卵巢、脾、大脑、肾、子宫、肺、肝和心脏。3种受体的表达,总体上来说在子宫中相对较高。TGFβ1及其受体在肾上腺和子宫等组织中有相对较高的表达量,提示其在繁殖过程中可能发挥作用。