侵染月季的李坏死环斑病毒的检测及CP基因序列分析

近年来，随着我国切花月季（Rosa hybrid）种植面积的不断增加，月季病毒病的发生日趋严重。据笔者调查，平均发病率20％～30％，有的品种如“绿宝石”，发病率超过60％。李坏死环斑病毒（Prunus necrotic ring spot virus，PNRSV）是月季的主要病毒之一。国内对月季病毒病的研究较少。为明确我国切花月季的主要病毒种类，加强对月季种苗质量的监督检测，笔者对侵染月季的PNRSV云南分离物（PNRSV—yunnan）进行了CP基因克隆及序列分析，建立了快速RT—PCR检测技术。     

PCR-based determination of colonization patterns during potato tuber infection by single and multiple pathogens

Potato tubers piled in storage are prone to infection by numerous pathogens. Each pathogen can cause damage alone, but severe losses often arise when more than one pathogen is involved. Currently, only a visual diagnosis is practiced on potato tubers before storing them, which does not allow any prediction of further disease spread. The aim of the present study was to determine differences in patterns of tissue colonization by several tuber decay pathogens and how late blight infection affects further tuber colonization by other important tuber pathogens. This study was conducted using artificial inoculation of potato tubers and PCR to provide an early and accurate diagnosis of disease development for major potato tuber rots, and to assess potential synergism/antagonism between Phytophthora infestans and other pathogens in stored tubers. In order to accurately follow the progress of each pathogen in tuber tissues, samples were collected over time from both the surface (peel, 0–2 mm depth) and internal tissues (flesh, depth > 2 mm) of the tubers at various distances from the inoculation site, at 3, 5, 7, 10, 12, 14, 17, and 19 days after inoculation. Successful detection of single or multiple pathogens was achieved using specific PCR-primers for each pathogen. Pathogens were always detected several centimeters ahead of the visible lesions. This tracking enabled us to determine the extent of colonization both on the tuber’s surface and in internal tissues by each tested pathogen, either after single or multiple infections involving P. infestans as the primary pathogen. The presence of P. infestans was shown to enhance the development of Pectobacterium atrosepticum and to slow down that of P. erythrospetica and Pythium ultimum. No noticeable effect on further tuber colonization by F. sambucinum, V. dahliae or V. albo-atrum was observed in the presence of P. infestans. This approach involving more than one pathogen is more realistic than classical studies considering single pathogens, and may be helpful in monitoring the sanitary status of stored tubers. Our results make the outcome of certain combinations of pathogens in potato tubers more predictable and may result in more efficient preventive measures.     

应用RT-PCR和斑点杂交法检测新疆梨树上的苹果锈果类病毒

利用自行设计的方法分离提取总RNA,通过一对特异引物,分别扩增出不同梨树品种上苹果锈果类病毒的特异片段,通过对库尔勒香梨品种上获得的特异片段回收、克隆和测序。结果表明,该片段长250bp,与已发表的AF421195同源性为99%,在此基础上利用克隆的ASSVd质粒,通过RT-PCR合成了生物素标记的cDNA探针,利用该探针对梨样品进行了斑点杂交检测,取得了很好的检测效果。进一步证明该反应体系能很好地用于梨树苹果锈果类病毒的RT-PCR检测。     

应用多重引物RT-PCR快速诊断禽流感亚型

将本实验室分离保存、已经过PCR扩增、核酸测序鉴定的三种不同亚型禽流感病毒(AIVH5N1、H6N2、H9N2)分别接种鸡胚,收集尿囊液,分离提取AIV总RNA。参考H1￣H15亚型禽流感病毒的核蛋白(NP)基因序列以及H5、H6、H9亚型AIV血凝素(HA)基因序列,设计四对引物:NP1,NP2;H5P1,H5P2;H6P1,H6P2;H9P1,H9P2,混合为PCR扩增多重引物MP1,下游引物NP2、H5P2、H6P2,、H9P2混合为逆转录反应多重引物MP2。AIV总RNA在相同反应条件下经MP2逆转录,用MP1进行PCR扩增,AIVH5亚型材料得到205bp,563bp两条扩增带,AIVH6亚型材料得到205bp,233bp两条扩增带,AIVH9亚型材料得到205bp,690bp两条扩增带,都与实验设计相符合,阴性材料没有扩增带。该方法成功实现四对引物进行RT-PCR反应同时检测出AIV,准确诊断H5、H6、H9三种禽流感亚型,检测时间短,特异性强,能及时检测出AIV亚型。     

TGEV细胞培养物超速离心与RT-PCR扩增的研究

研究猪传染性胃肠炎病毒(TGEV)细胞培养物的超速离心及鉴定方法.将TGEV的细胞培养物用饱和硫酸氨法浓缩后,用200 g/kg～500 g/kg蔗糖密度梯度离心分装,测定蛋白浓度并取峰值管进行RT-PCR,并设立15、25、35三个不同循环参数测定PCR产物volume值.结果表明,在35个循环条件下第14号样品PCR产物的volume值最大,表明该样品病毒核酸的含量最大,由此推算病毒粒子主要集中在360 g/kg～380 g/kg蔗糖区带处,这与理论值相符,证明猪传染性胃肠炎病毒经密度区带离心后,利用PCR产物量确定目的病毒区带效果显著.     

Reversal effect of FG020318 on multidrug resistance in retinoblastoma cell line SO-Rb50

AIM: To investigate the multidrug resistance (MDR),reversal activity of 2-［4-(2-pyridin-2-yl-vinyl) henyl］-4,5-bis-(4-N,N-diethylaminophenyl)-1 (H)-imidazole (FG020318) in a retinoblastoma subline SO-Rb50/VCR,which is resistant to vincristine.METHODS: The procedure of stepwise increase in drug concentrations was used to obtain SO-Rb50/VCR,which was resistant to 200 μg/L vincristine.The chemosensitivity of this drug resistant cell line with and without FG020318 or cyclosporine A (CSA) were detected by MTT assay and the function of p-glycoprotein (P-gp) was examined by rhodamine 123 accumulation detected with flow cytometry (FCM).RESULTS: FG020318 (2.5 μmol/L) significantly reduced IC50 and increased the rhodamine accumulation in a concentration-dependent manner.It was much stronger than the positive control CSA in reversal of MDR.CONCLUSION: A new tumor MDR modulator FG020318 partly reverses MDR in SO-Rb50/VCR.It may be a promising new drug to tackling MDR.     

沙地葡萄茎痘相关病毒RT-PCR检测

为建立快速、灵敏、可靠的沙地葡萄茎痘相关病毒(GRSPaV)检测方法,以总RNA为模板,采用2组GRSPaV特异性引物对29个品种52株葡萄样品进行RT-PCR检测,并对扩增产物进行了测序和分析。结果表明,从20个品种25株葡萄样品中检测到GRSPaV,平均带毒株率为48.1%。外壳蛋白基因片段引物F1/R1从25个样品中扩增到905bp的特异片段,复制酶基因片段引物F2/R2从20个样品中扩增到498bp的特异片段,表明GRSPaV外壳蛋白基因比复制酶基因更加保守,RT-PCR检测时采用F1/R1则更为适宜。PCR产物测序结果与GenBank中登录的GRSPaV序列比较,同源性为97.90%～98.11%。     

辣椒组织总RNA提取方法研究

从RNA的完整性、纯度和产量等方面比较了3种RNA提取试剂对辣椒不同组织部位RNA的提取效果。结果表明,RNAplant能从成熟组织中获得较高质量和产量的RNA,用该方法提取的辣椒老化组织RNA经RT-PCR能成功进行内参检测。     

朊蛋白基因在奶牛生殖系统不同组织中的表达量测定

为了研究疯牛病能够垂直传播的理论基础,我们采用PrP基因做目的基因,GAPDH基因做内参照的相对荧光定量RT-PCR方法,对目的基因和内参基因分别构建标准重组质粒,制备标准曲线,利用标准曲线对采集雌雄奶牛生殖系统的12个不同组织PrP基因表达进行定量。试验结果表明雌雄奶牛的生殖系统各部分组织都有PrP基因表达,其中雄性奶牛的睾丸、附睾和输精管呈现高的表达量,分别为1.16±0.0010、1.81±0.0056、0.68±0.0038;前列腺、精囊腺和尿道球腺呈现中等表达量,分别为0.47±0.0018、0.25±0.0007、0.28±0.0007;阴茎呈现低的表达量为0.18±0.0017;雌性奶牛的子宫、子宫阜呈现高的表达量,分别为2.53±0.0008、1.75±0.0098;卵巢和输卵管呈现中等表达量,分别为1.55±0.0038、1.45±0.0064;阴道呈现低表达量为0.57±0.0036。本研究为阐释PrP基因在奶牛生殖系统的正常表达和正常的生理功能,以及为确定生殖系统在疯牛病垂直传播中的地位和其分子机理奠定了基础。