鸡传染性支气管炎病毒地高辛标记探针检测方法的的建立和应用

根据GenBank中已经发表的IBV N基因的保守序列,设计合成一对引物,利用RT-PCR技术扩增IBVN基因的582 bp的核酸片段,并制备出地高辛标记的IBV核酸探针。特异性检测结果表明,该探针能与不同毒株的IBV核酸发生特异性杂交,而与对照的NDV、鹅副粘病毒的核酸杂交反应为阴性;敏感性检测结果表明,该探针对IBV的最低检出量为10 pg,显示所制备的核酸探针用于IBV的检测是可行的。     

鸡血管活性肠肽RNA探针制备及其在鸡肠Remak神经的原位杂交反应

应用RT-PCR方法从鸡脑中扩增血管活性肠肽(VIP)基因片段,将其连接于pGM-T easy.分别利用pGM-T easy中T7和SP6启动子及其RNA聚合酶,以线性化的VIP/pGM-T easy为模板转录合成正、反义DIG标记RNA探针.通过原位杂交方法将合成的探针用于探查VIP-mRNA在鸡肠Remak神经(INR)中的分布情况.结果表明,INR的神经元胞体中有VIP-mRNA的转录,且这种神经元数量众多;原位杂交阳性神经元胞体大部分是大型神经元细胞,呈圆形或椭圆形;阳性神经元胞体在INR神经节中呈层状或成群分布.INR的神经纤维呈弱阳性.在节间束也有少量的阳性细胞分布.本研究从基因水平证明INR中有VIP神经元存在.     

Quantitation of RNA tumor viruses and viruslike particles in human milk by hybridization to polyadenylic acid sequences

RNA tumor viruses and viruslike particles from human milk are quantitated by hybridization of the polyadenylic acid regions in their 60S to 70S RNA to radioactive polyribouridylic acid of known specific activity. The length of the polyadenylic acid region in the 60S to 70S RNA of the human milk particle is identical to that of the known oncogenic RNA viruses.     

动植物总核酸提取试验

依据最简单的核酸提取3步骤:细胞裂解、去除杂质、沉淀核酸的原理设计了一种普适性的动植物核酸提取方法(简称PS法),并对包括裸子植物门的银杏纲、苏铁纲、松柏纲,被子植物门的双子叶植物纲和单子叶植物纲的20种植物材料以及动物界的5纲5种材料进行了提取试验.结果表明,用PS方法提取动植物总核酸均能取得较理想的提取效果.所提取的总核酸分别用RNase和DNase进行消化,可分离纯化出高纯度的DNA和RNA.     

Use of phosphorus-32 in microassay for nucleic acid synthesis in Escherichia coli

A method was developed to facilitate studies of ribonucleic acid and deoxyribonucleic acid synthesis in growing bacterial cultures of small volume. The nucleic acid fractions were separated by means of a modified Schmidt-Thannhauser procedure applied to micro quantities of P(32)-labeled cells collected on membrane filters. The validity and usefulness of the method are discussed.     

鲑鱼甲病毒阳性核酸物质制备及RT-PCR检测方法的建立

为建立快速检测鲑鱼甲病毒(Salmonid alphavirus,SAV)RT-PCR方法,根据Gen Bank公布的E2基因序列(1 375 bp)设计引物,扩增S AV E2全长基因,利用假病毒制备技术获得包裹E2核酸物质(RNA)的SAV假病毒溶液。以SAV假病毒作为阳性核酸物质质控品,以E2F:5'CCG-TTG-CGG-CCA-CAC-TGG-ATG 3',E2R:5'CCT-CAT-AGG-TGA-TCG-ACG-GCA-G 3'为引物,优化反应条件,建立SAV的RT-PCR检测方法。结果表明,该方法可扩增SAV E2特异性516 bp的DNA片段,引物最适反应浓度为1.0μmol·L-1,最佳退火温度为57.5℃,对SAV的三种亚型SAV1(V4640)、SAV2(V4619)、SAV5(V4638)检测结果均为阳性,对SVCV、IHNV和IPNV的PCR扩增结果均为阴性;并确定对SAV的核酸最低检出量达1.59 pg;以RT-PCR方法在不同时间对每份样品作3次重复检测,检测结果一致。表明此方法特异性强,敏感性高,稳定性和重复性较好,可用于SAV临床诊断和检测。     

新冠病毒核酸检测等待时间缩短策略研究—基于品管圈的应用

目的 观察品管圈对缩短新冠病毒核酸检测等待时间的效果.方法 选取2020年7月品管圈改善前新冠病毒核酸检测者247名(对照组)进行调查,分析影响核酸检测等待的主要因素,制定出相应的对策;选取2021年3月新冠病毒核酸检测者251名(观察组)实施经品管圈改善后制定的对策.对比两组受检者采样前等待时间、就诊知晓率和服务满意...     

An optimized protocol using Steedman's wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber

In situ mRNA hybridization (ISH) is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber. The most common ISH protocol uses paraffin wax; however, embedding tissue in paraffin wax can take a long time and might result in RNA degradation and decreased signals. Here, we developed an optimized protocol to simplify the process and improve RNA sensitivity. We combined embedding tissue in low melting-point Steedman's wax with processing tissue sections in solution, as in the whole-mount ISH method in the optimized protocol. Using the optimized protocol, we examined the expression patterns of the CLAVATA3 (CLV3) and WUSCHEL (WUS) genes in shoot apical meristems and floral meristems of Cucumis sativus (cucumber) and Arabidopsis thaliana (Arabidopsis). The optimized protocol saved 4–5 days of experimental period compared with the standard ISH protocol using paraffin wax. Moreover, the optimized protocol achieved high signal sensitivity. The optimized protocol was successful for both cucumber and Arabidopsis, which indicates it might have general applicability to most plants.