The Value of Tissue Imprint Hybridization for Rapid Detection of Infectious Bursal Disease Virus from Field Outbreaks

The use of a peroxidase labelled PCR generated probe followed by enhanced chemiluminescence hybridization assay detected infectious bursal disease virus directly from bursal imprints on a nylon membrane. Tissue imprint hybridization proved to be a simple, rapid and safe means of detecting IBD virus for screening large numbers of field samples. The PCR generated probe was highly specific for IBD virus and did not hybridize with cellular nucleic acids in control imprints. Tissue imprint hybridization was found to be a more sensitive method than conventional antigen detection assays.     

Double in situ hybridization for simultaneous detection and differentiation of porcine circovirus 1 and 2 in pigs with postweaning multisystemic wasting syndrome

Double in situ hybridization using a digoxigenin-labelled porcine circovirus 1 (PCV1) and biotinylated PCV2 probe, was developed for the simultaneous detection and differentiation of PCV1 and PCV2 in formalin-fixed, paraffin-embedded tissues from pigs with postweaning multisystemic wasting syndrome. The combination of an alkaline phosphatase conjugated antidigoxigenin system with alkaline phosphatase conjugated streptavidin-biotin system allowed identification of PCV1 and/or PCV2. No evidence of cross-reaction was observed. Positive cells exhibited a red or dark brown reaction product for PCV1 and PCV2, respectively. Both PCV DNAs were observed mainly in the cytoplasm but occasionally in the nucleus. Co-localization of hybridization signal for both PCV1 and PCV2 was present in macrophages and multinucleated giant cells of the lymph node and spleen. This double-labelling technique for the differentiation between PCV1 and PCV2 is suitable for pathogenesis studies and diagnostic applications.     

一个山羊Ⅰ型毛角蛋白基因的序列及其在皮肤中的表达

角蛋白家族包含表皮软角蛋白和毛发硬角蛋白，可以分为酸性Ⅰ型和碱性或中性的Ⅱ型角蛋白亚家族。从构建的成年山羊皮肤cDNA文库和ESTs分析中发现了和与绵羊羊毛角蛋白8C1和人Ⅰ型毛发角蛋白相应cDNA序列高度同源的序列，经序列分析证明是一个山羊毛发特异性角蛋白，命名为山羊Ⅰ型毛角蛋白1（gHa1），Gen Bank序列号为AY510110．1。推导的读码框ORF由413氨基酸组成，与绵羊8C1角蛋白的序列一致性为97．8％。原位杂交显示它在皮肤初级和次级毛囊的皮质层有强烈的表达。     

成年发情期奶山羊下丘脑催产素及其mRNA的表达

应用免疫组织化学SP法和原位杂交法研究了催产素（oxytocin，OT）及OT mRNA在成年发情期奶山羊下丘脑中的分布和表达。结果，OT免疫反应阳性细胞主要分布在视上核和室旁核，在视上弥散核、弓状核、室周核和乳头体各核团也存在免疫阳性神经元；在室旁核、视上弥散核、正中隆起和第三脑室附近有较多数量的强阳性神经纤维，在交叉上核有少量阳性神经纤维。在下丘脑23个核团（区）中均能检测出OT mRNA的阳性细胞。结果表明，OT和OT mRNA在下丘脑中分布广泛，且OT可能通过轴突传递和血液运输，将OT mRNA合成的OT运送到别的核团；OT在奶山羊发情过程中发挥了重要作用。     

Phenotypic and histological expression of different genetic backgrounds in interactions between lettuce, wild Lactuca spp., L. sativa × L. serriola hybrids and Bremia lactucae

Phenotypic and histological responses of cultivated lettuce (Lactuca sativa) and wild relatives L. saligna, L.␣virosa as well as interspecific crosses derived from L. sativa × L. serriola to two races of Bremia lactucae (CS2, CS9) were investigated. With the exception of L. sativa genotypes, all accessions and hybrids expressed incomplete or complete resistance to both pathogen races, with slight differences at seedling and adult plant stages, respectively. Histological features of the interactions (development of pathogen infection structures and host hypersensitive response to attempted infection) were studied on leaf discs 48 h after inoculation. Interactions with similar phenotypic expression of resistance were characterized by significant variation in rate of development of pathogen infection structures and hypersensitive reactions. Differences found within eight Lactuca spp. accessions and hybrids challenged by two distinct pathogen races are interpreted and discussed.     

地高辛标记cDNA探针检测苹果茎痘病毒

 Partial sequence(314 bp) of ASPV was cloned and used as a probe labelled with digoxigenin-11dUTP. The total RNA extracted from samples with Apple stem pitting virus and a series of dilutions of plasmid with ASPV-cDNA were detected by dot blot hybridization. The results showed that the probe was sensitive and specific. The probe couldn't hybridize with total RNA of Apple stem grooving virus, Apple mosaic virus and Apple chlorotic leaf spot virus samples as well as negative control, only hybridized with that extracted from dormant shoot infected with ASPV. The sensitivity for detection of plasmid contained ASPV-cDNA was 1.64 μg.     

猪附红细胞体PCR-微孔板杂交-酶免疫检测方法的研究

以猪附红细胞体和多种血营养菌16S rRNA基因的保守区设计合成引物HM-f/HM-r,在反向引物的5′端用生物素标记,以猪附红细胞体广东株16S rRNA基因的可变区序列设计种特异性寡核苷酸探针,探针的5′端用地高辛标记,成功地建立了猪附红细胞体PCR-微孔板杂交-酶免疫检测方法。通过测定不同浓度PCR产物对应的微孔板杂交-酶反应显色OD值,用SPSS统计软件进行回归分析,得到回归方程为y=2.1012-0.4492×logx,其相关系数R为0.977,显示对未知样品可进行定量检测。该方法与猪的多种细菌性病原、猫血巴尔通氏体CA株和E.wenyonii无交叉反应,其敏感性比常规PCR琼脂糖电泳检测提高了近100倍。用建立的方法对38份临床样品进行检测,结果有12份检测结果为阳性。     

猪蛔虫感染期幼虫抑制消减cDNA文库的构建

为筛选与线虫感染性相关的基因,本研究以猪蛔虫为对象,构建猪蛔虫感染期幼虫差异表达消减cDNA文库,为研究线虫期特异性发育的分子机制奠定基础。分别提取感染期幼虫和其它各期幼虫及成虫的总RNA,纯化mRNA后,采用Clontech公司PCR-selectTM试剂盒进行反转录合成cDNA并进行抑制消减杂交(SSH),构建猪蛔虫感染期幼虫差异表达的消减cDNA文库,并采用Southern斑点杂交进行消减效率的检测。随机从文库中抽取45个克隆进行测序及在线BLAST分析。试验结果表明,感染期幼虫差异表达的消减cDNA文库具有较强的特异性;在得到的41个表达序列标签(ESTs)中,有40个ESTs与已报道的基因有较高的相似性,主要代表猪蛔虫第三期幼虫基因和成虫头部基因,有1个cDNA片段可能代表新基因。猪蛔虫感染期幼虫差异表达消减cDNA文库的成功构建,为进一步研究幼虫发育差异表达基因的功能奠定了基础。     

Induction of interspecific allo-tetraploids of Megalobrama amblycephala♀×Megalobrama terminalis♂ by heat shock

Interspecific allo-tetraploidy was induced by a combination of hybridization of Megalobrama amblycephala ♀× Megalobrama terminalis ♂ and thermal shock. A thermal shock at 40 °C for 2 min applied to eggs 33 or 26 min (τ₀: 1.40) after fertilization resulted in a significantly ( P <0.01) higher yield of allo-tetraploids than hybrid crosses without heat shock. The yield of allo-tetraploids in embryos from mid-gastrula to hatching stages was 15.7–24.4% in different thermal shock treatment groups. The allo-tetraploidy rate in 330-day-old juveniles was 9.3% and 6.7% in different year treatment groups. After 2 years of rearing, allo-tetraploidy males attained sexual maturity with milk-like milt at the age of 2+, while allo-tetraploidy females at the age of 2+, and even at 3+, did not show any signs of sexual maturation exhibiting obviously delayed maturity. Only a few allo-tetraploidy females (three individuals) showed maturation characters at the age of 4+ and these females were induced to spawn.     

中华鳖脾脏雄激素受体mRNA 的原位杂交检测

采用原位杂交技术（In situ hybridization，ISH）研究雄激素受体（Androgen receptor，AR）mRNA在中华鳖（Pelodiseus Sinensis）脾脏的存在及细胞定位。取5只健康成年中华鳖的脾脏，进行石蜡切片。用地高辛标记的寡核苷酸探针对组织切片进行原位杂交（In situ hybridization，ISH），检测雄激素受体mRNA在脾脏免疫细胞内的表达定位。结果显示，大部分雄激素受体mRNA阳性细胞呈圆形或椭圆形，数量少胞体大且呈不规则形。阳性细胞在脾脏动脉周围淋巴鞘（Pefiatefial lymphatic sheath，PALS）和红髓（Red pulp，RP）内分布密集。椭球周围淋巴鞘（Periellipsoidal lymphatic sheath，PELS）内AR mRNA阳性细胞分布极少。杂交阳性信号物质在细胞质和细胞核均有分布。在标记为阳性的中华鳖脾脏中，推测圆形的阳性细胞可能为B淋巴细胞，而另一部分胞体大且形状不规则的阳性细胞则可能为巨噬细胞。脾脏内AR mRNA的存在进一步证实雄激素可能是调节脾脏等免疫器官功能的因素之一。[中国水产科学，2008，15（2）：337—341]