猪流行性腹泻病毒RT-PCR检测方法建立及初步应用研究

为建立一种特异、敏感的PEDV的检测方法。根据GenBank中公布的猪流行性腹泻病毒CV777株的M基因序列,设计了一对特异性引物,扩增长度为680 bp的片段。将PCR产物进行测序,与CV777株、SH5株M基因的同源性分别为99.2%和97.6%。试验表明：该方法具有较好的特异性和敏感性,并能检测到PEDV的RNA浓度下限为100 pg/μL。从福建省的3个地区共采集58份哺乳仔猪腹泻样品,并应用此方法进行检测,其阳性检出率为87.9%。     

恶疫霉实时定量RT-PCR分析中内参基因的选择

为选择合适的内参基因用于分析植物病原卵菌恶疫霉(Phytophthora cactorum)的基因表达情况,本研究利用实时定量RT-PCR分析持家基因Ubc、Tub-b、WS21和WS41在恶疫霉生长发育阶段和侵染草莓阶段的表达差异,并利用软件geNorm、NormFinder和BestKeeper比较其表达稳定性。结果表明：基于公共数据库序列和其他疫霉菌同源序列设计的持家基因引物在恶疫霉中具有良好的扩增效率、反应有效性和特异性。通过分析,发现在恶疫霉的不同生长发育阶段（菌丝生长、游动孢子囊、游动孢子和休止孢萌发）和侵染草莓阶段（接种后3、5、7天）,表现最为稳定的是Ubc基因。当使用多个内参基因时,Ubc和WS41是最适合校正恶疫霉基因表达数据的内参基因组合。本研究结果为利用实时定量RT-PCR准确分析恶疫霉基因相对表达量提供了重要的内参基因,也为其他疫霉菌的基因表达研究提供了有价值的参考。     

着床前小鼠孤雌囊胚及正常囊胚上Wnt-3a的差异表达研究

为了研究Wnt-3a在小鼠着床前,孤雌囊胚和正常胚胎上的表达差异情况。采用激光扫描共聚焦显微技术和半定量RT-PCR检测方法,对孤雌囊胚和正常囊胚上Wnt-3a的表达,以及分布情况进行定性和定量的检测。利用激光扫描共聚焦显微技术检测发现：在着床前的小鼠孤雌囊胚和正常囊胚上都有Wnt-3a蛋白质的表达,且只在滋养层细胞中表达。通过半定量RT-PCR检测发现,着床前小鼠孤雌胚胎与正常胚胎相比,Wnt-3a的转录水平没有显著性差异。所以Wnt-3a在着床前小鼠孤雌胚胎和正常胚胎中都有表达,而且其转录水平在这2种胚胎中没有显著性差异。     

水仙花叶病毒RT-PCR检测方法的建立及应用

旨在建立水仙花叶病毒(Narcissus mosaic virus,NMV)的分子快速检测方法。根据已公布的NMV基因序列,设计一对特异性引物,以提取的水仙样品总RNA为模板进行RT-PCR,并对该方法的特异性、灵敏度、重复性及实际检测效果进行测定。从感染NMV的水仙样品中成功扩增出预期大小（约483 kb）的特异性目的片段,而健康水仙上未扩增到相应的片段。特异性、灵敏度及重复性测定结果表明,本研究建立的RT-PCR方法特异性强、灵敏高、重复性好。应用该方法成功检测了一批口岸送检的水仙样品。本研究建立的RT-PCR方法可为水仙上NMV的检测提供可靠的技术支持。     

应用常规RT-PCR和荧光定量RT-PCR检测柑桔衰退病毒

 根据柑桔衰退病毒(CTV)P20基因序列设计cquctv9/cquctv10特异引物对,以柑桔RNApolymeraseⅡ基因作为内参照,建立了柑桔衰退病的常规RT-PCR快速检测体系;依据柑桔衰退病毒P20基因序列设计cquctv1/cquctv2特异引物对和TaqMan探针cquctvp1,建立了柑桔衰退病荧光定量RT-PCR快速检测体系。常规RT-PCR检测下限是含有CTV的50pg总RNA,荧光定量RT-PCR法的检测下限是2fg纯CTV片段。荧光定量RT-PCR的灵敏度相对比常规RT-PCR高100倍。利用常规RT-PCR和荧光定量RT-PCR体系对从2005年3月到2006年7月采自田间的样品进行检测,结果表明,2种检测体系都有很好的特异性和准确性;对183个田间柑桔苗木样品带毒率检测结果表明,荧光定量RT-PCR检出率为(82.5%),比常规RT-PCR检出率(73.2%)高。     

应用MNP-RT-PCR方法检测黄瓜绿斑驳花叶病毒

 A novel RT-PCR method integrated with Magnetic Nano Particles (MNP), MNP-RT-PCR, was set up for detection of Cucumber green mottle mosaic virus (CGMMV). After the virus particles in crude sap were concentrated by MNP, viral RNAs were released and were detected by RT-PCR. CGMMV could be detected in as less as 10 ng watermelon leaf materials. Compared with normal RT-PCR, the method decreased the inhibitors of plant material and steps for extracting RNA, and also increased the sensitivity of RT-PCR detection in less time. The method is simple and suitable for quick detection of plant virus in a large number of samples.     

Incidence and genetic diversity of Peach latent mosaic viroid and Hop stunt viroid in stone fruits in Serbia

Tissue-imprint hybridization (TIH) assay was validated for large-scale detection of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). All 72 collected leaves (100%) from 2 PLMVd- and 2 HSVd-infected trees were positive in TIH, regardless of the geographic orientation of the scaffold, level of the canopy and position of the leaf in the shoot. In a large-scale survey in Serbia, we tested by TIH 871 trees of stone fruits, representing 602 cultivars from fruit collections in Belgrade, Čačak and Novi Sad. PLMVd was detected in 185 (50%) peach trees or 95 (54%) cultivars and HSVd in 2 apricot trees and cultivars (2%). The occurrence of HSVd is a new report for Serbia. No viroid infection was found in European plums, sweet cherries, sour cherries and wild Prunus spp. PLMVd-infected peach cultivars originated from the world’s main breeding centres of this crop. Western European and Asian cultivars were the most infected (58%) followed by those originating from North America (50%). Nine PLMVd and two HSVd isolates were sequenced and analyzed. All showed PMLVd sequences clustered together in the previously reported phylogenetic group III. Both HSVd isolates were found to be derived from recombinant events, but that of the cv. Saturn represented a putative new phylogenetic group of HSVd.     

进境日本豇豆中黑眼豇豆花叶病毒的检测与鉴定

从进口日本豇豆中随机挑选种子在隔离温室内种植。出苗后,发现有典型花叶症状的植株。对该病株进行电镜观测,发现有线条形病毒粒体,大小约为750nm×13nm。设计特异性引物(BL-5/BL-6),对该样品进行黑眼豇豆花叶病毒的反转录(RT)PCR分子检测。电泳检测表明,扩增出一条特异性的目标条带,大小为338bp。对该样品的RT-PCR产物进行测序,结果表明该序列与黑眼豇豆花叶病毒(GENEBANK登录号:AY575773)的序列有4个碱基差异,同源性为98.8%。鉴定该病毒为黑眼豇豆花叶病毒(Blackeye cowpea mosaicvirus)。