猪皮炎肾病综合征组织病理学和PCR诊断

应用病理学和PCR方法,对浙江某猪场送检的疑似猪皮炎肾病综合征（PDNS）病猪进行诊断。结果发现肾脏呈现纤维蛋白性肾小球肾炎变化,真皮及皮下血管表现为坏死性脉管炎,淋巴组织中大量淋巴细胞缺失、多核巨细胞浸润,PCR扩增产物显示猪圆环病毒2型（PCV-2）阳性。结果表明,该猪场感染了PCV-2,并表现为PDNS的病理特征。     

杂草稻nrDNA ITS片段的PCR扩增条件优化及测定

对杂草稻的nrDNA ITS片段的PCR扩增条件进行了优化并测定,建立的PCR最优体系为：20μL反应体系含1μL模板DNA,0．125mmoL／L dNTPs,0．5μmol／L正反向引物,1U Taq酶,2．0μL 10×Taq PCR Bufter;退火温度为57℃。这样的条件下充分保证了ITS PCR产物的质量和纯度要求,直接测序结果为600bp左右,与网上结果十分类似,表明结果准确可靠。这些ITS片段的系统学信息将为杂草稻的起源进化提供有力的分子水平证据。     

苦瓜MAP30基因的克隆与载体构建

首先提取苦瓜的基因组DNA,然后根据Genbank中已公开发表的MAP30序列,设计一对特异引物MAP301,MAP302,并增添特异定位于内质网的小肽Kozak序列。采用PCR技术,从苦瓜的基因组DNA中扩增出一分子量为800bp左右的片段,回收克隆测序,结果表明,克隆获得片段与已公开发表的MAP30序列同源性达100%,将阳性克隆进行酶切,获得的目标片段与用同样酶切所获得的植物表达载体pEV1和pEV2大片段连接,酶切鉴定重组子构建成特异表达于果实的载体。     

应用多重荧光PCR快速筛查作物中转基因成分研究

通过对我国批准进口的和获得农业转基因生物安全证书的转基因玉米转化体的序列分析发现,除DAS40278-9和BLVA430101外,CaMV35s启动子、NOS终止子、CrylAb/Ac基因和pat基因覆盖了 21种玉米转基因转化体.通过引物组合筛选、反应体系优化、灵敏度测试、适用性测试等实验,建立了基于4个通用筛选元件...     

Isolation and pathogenicity of Colletotrichum spp. causing anthracnose of strawberry in south west Spain

Anthracnose, caused by Colletotrichum spp., is a major disease of cultivated strawberry, Fragaria × ananassa. This study identifies the Colletotrichum spp. which causes strawberry anthracnose in the southwest of Spain. A survey of the region was carried out, and the strains isolated were identified as C. acutatum by using the polymerase chain reaction (PCR) with genus and species-specific primers, demonstrating that this species is currently the causal agent of strawberry anthracnose in the studied region. The pathogenicity of C. acutatum and C. gloeosporioides strains was evaluated on two principal strawberry cultivars (cvs Camarosa and Ventana) under field conditions, the latter being more pathogenic than the former.     

Use of real-time PCR to examine the relationship between disease severity in pea and <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis> DNA content in roots

Aphanomyces euteiches causes severe root rot of peas. Resistance is limited in commercial pea cultivars. Real-time fluorescent PCR assay specific for A. euteiches was used to study the relationship between disease severity and pathogen DNA content in infected peas. Five pea genotypes ranging in levels of resistance were inoculated with five isolates of A. euteiches. Plants were visually rated for disease development and the amount of pathogen DNA in roots was determined using the PCR assay. The susceptible genotypes Genie, DSP and Bolero tended to have significantly more disease and more pathogen DNA than the resistant genotypes 90-2079 and PI 180693. PI 180693 consistently had less disease, while 90-2079 had the lowest amount of pathogen DNA. The Spearman correlation between pathogen DNA quantity and disease development was positive and significant (P < 0.05) for three isolates, but was not significant for two other isolates. This suggests that the real-time PCR assay may have limited application as a selection tool for resistance in pea to A. euteiches. Its utility as a selection tool would be dependent on the correlation between disease development and pathogen DNA content for a given pathogen isolate. The accuracy and specificity of the real-time PCR assay suggests considerable application for the assay in the study of mechanisms of disease resistance and the study of microbial population dynamics in plants.     

龙眼体胚发生早期miR166初级体的克隆与表达分析

为了解龙眼miR166初级体基因Pri-miR166 S53结构特点及其前体和成熟体在龙眼体胚发生早期的表达模式,采用SMART~(TM) RACE试剂盒和PCR扩增技术克隆龙眼体胚早期miR166基因的初级体(Pri-miR166 S53),确认其转录起始位点,并预测其含有的潜在ORF;利用龙眼基因组数据库提取其启动子序列,预测其含有的顺式作用元件;用实时荧光定量PCR对龙眼体胚发生早期及不同激素处理下的胚性愈伤组织中miR166基因的前体(Pre-miR166 S53)和成熟体(miR166a.2)表达模式进行分析。结果表明:获得长317 bp的Pri-miR166 S53基因初级体序列,对其进行翻译,得到一条长13个氨基酸序列的miPEP(MLCFVDALFLIST)。利用生物信息学软件分析Pri-miR166 S53基因的启动子序列发现,除了具有TATA/CAAT-box外,还含有生长素、脱落酸、乙烯、水杨酸、茉莉酸甲酯及spl、HSE等特异作用元件。实时荧光定量PCR分析表明,在2,4-D调控的龙眼体胚发生早期过程中,从胚性松散型愈伤组织发育到球形胚的过程中,Pri-miR166 S53基因的前体pre-miR166 S53和成熟体miR166a.2都表现为下调趋势;而在无2,4-D调控的龙眼体胚发生早期中,pre-miR166 S53和miR166a.2表达模式不同。此外,pre-miR166S53随ABA和乙烯处理浓度升高呈下调表达,而对不同浓度2,4-D处理无应答;miR166a.2随2,4-D、ABA处理浓度升高呈下调表达,而在乙烯处理下呈上调表达。上述研究结果提示miR166前体和成熟体在对外源激素应答的模式上并不呈简单线性相关,可能存在多层次、多方位的调控。     

单管实时荧光RT PCR方法同时检测大豆种子中的菜豆荚斑驳病毒和烟草环斑病毒

 本研究建立了大豆种子中菜豆荚斑驳病毒（BPMV）和烟草环斑病毒（TRSV）单管双重实时荧光PCR检测方法。将含有相同浓度的分别带有BPMV和TRSV CP基因的质粒溶液作为阳性对照,以受两种病毒侵染的大豆种子作为待测样品进行实时荧光PCR检测,结果表明能从同一管中同时检测出这两种病毒而不发生交叉反应。尽管在阳性对照中,二者的检测限相当,均可达到35 pg/mL,但在实际应用中,两种病毒由于在大豆种子中的浓度不一致而存在一定的差别。该方法快速、灵敏、简便,同时特异性更强,在出入境检验检疫中具有广泛的应用前景。     

PCR检测猪输血传播病毒Ⅰ型方法的建立及其初步应用

参照GenBank已公布的猪源输血传播病毒Ⅰ型（Torque Teno Virus genogroupⅠ,TTVⅠ）全基因序列,设计并合成一对特异性引物,PCR扩增片段为194bp,测序结果与已公布的猪TTVⅠ型序列同源性为92.6%。用所建立的方法检测猪圆环病毒Ⅱ型、猪细小病毒、猪伪狂犬病病毒、猪多杀性巴氏杆菌、猪链...