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本实验采用一种非放射性物质——碱性磷酸酶标记乙肝病毒HBV DNA制备分子探针。碱性磷酸酶在苯醌作用下与单链DNA联结,形成DNA和酶的共价复合物,即酶标探针。此探针通过分子杂交与待测DNA结合,与酶的底物作用显色,几小时内可观察结果,其最低检测量约为10pg。用此探针检测乙肝病人血清中的HBV DNA,与~(32)P标记的探针比较,酶标探针可检测出~(32)P标记探针检出率的95.7%。结果表明,所合成的酶标探针具有准确、简便、快速、安全而经济的优点,具有应用前景。 相似文献
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使用国产辣根过氧化物酶(HRP)合成酶标记物,用竞争法进行了植物内源激素脱落酸(ABA)的酶标免疫测定研究。测定范围为0.0125ng—100ng,在此范围内logitB/B。与ABA浓度的对数之间呈较好的线性关系。检测的灵敏度达到5×10~(-14)克分子。比较了两种酶标记方法对于测定的影响,结果发现直接使ABA共价结合到HRP上形成ABA-HRP酶标记物比先将ABA与牛血清白蛋白(BSA)结合后,然后进一步再与HRP反应形成ABA—BSA—HRP复合物灵敏度高,非特异性吸附小,而且合成步骤较少。酶标记物的稀释度直接影响测定的灵敏度和浓度对数与logit B/B。之间的线性关系好坏;在以每毫升3—6微克免疫球蛋白包埋免疫吸附板进行测定时,用每毫升5微克酶标记物的浓度获得了最佳结果。 相似文献
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李敏 《中国生物工程杂志》1989,9(6):38-41
DNA文库的建立及其应用于基因定位的可能性首先在基因组较小的生物(如果蝇)中得到证实。近年来原位噬菌斑和克隆杂交,改进的λ克隆载体以及体外包装系统等的发展,对于较复杂的基因组(哺乳动物)也进行了基因文库的建立和筛选。对复杂基因组中富集某一特定部分的文库的建立开始于七十年代末,被克隆的特定染色体DNA顺序文库代表全部基因组信息的结构亚单位,比整个基因组定位具有显著的优点。 相似文献
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本文建立了牛微量白细胞样品的处理及其Y染色体特异DNA的扩增的方法。采集成年公牛全血,用EDTA抗凝血。分离白细胞,用0.145mol/LNaCl洗净,用0.145mol/LNaCl稀释,计数。分别将0.5、50、500细胞在含10mmol/LTris-HCl、50mmol/LKCl、2mmol/LMgCl2、0.45%NP-40、0.45%Tween-20、0.1mg/mL蛋白酶K、总体积20μ 相似文献
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生物素标记贾第虫全基因组DNA探针的制备及其特异性敏感性测定 总被引:3,自引:0,他引:3
用生物素标记的贾第虫全基因组DNA探针,在斑点杂交试验中显示高度的敏感性和特异性。用它可检出10ng贾第虫DA,10^3个贾第虫滋养体或包囊,且不与阴道毛滴虫、溶组织内阿米巴、弓形虫和BABL/c小鼠肝细胞DNA,以及贾第虫患者粪便上清液发生交叉反应。本探针可用于贾第虫病病原体检测和虫株鉴定研究。 相似文献
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两种DNA探针杂交检测结核分支杆菌方法的研究 总被引:3,自引:0,他引:3
为改进结核杆菌DNA探针的特异性与实用性,研制了以生物素标记的两种对结核分支杆菌特异的DNA探针:一个5’端标记的20bp的寡核苷酸探针和一个采用PCR方法合成的188bp长链探针。两种探针分别与结核分支杆菌的全染色体DNA,以及基因组上IS6110序列的一段317bp的PCR扩增产物进行斑点杂交,以碱性磷酸酶(AP)催化的染色反应检测,测试了两个探针的敏感性和特异性。系统地比较研究了两种探针杂交检测条件:探针的浓度选择,杂交温度与洗膜温度的选择,以及杂交与洗膜温度对检测的敏感性与特异性的影响。寡核苷酸探针和188bp探针杂交检测纯化结核分支杆菌基因组DNA的敏感性分别为100ng与6ng,杂交检测PCR产物的敏感性分别是400pg与50pg。两探针的最佳杂交浓度均为40~160ng/ml,最佳杂交温度分别是42℃与68℃,最佳洗膜温度分别是60℃与60~68℃之间。两种探针均仅与结核分支杆菌及BCG有杂交信号,而与其它受试分支杆菌及非分支杆菌杂交结果都呈阴性。它们的特异性都很强,但188bp探针的敏感性约是寡核苷酸探针的7~16倍,而且188bp探针检测本底较低,是检测结核分支杆菌的较佳选择 相似文献
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大熊猫基因指纹探针F2ZGP96060801的研制及比较实验分析 总被引:4,自引:1,他引:4
利用ABI-394型DNA合成仪合成的寡核苷酸5-「A(X)n-xTCCAC」n-3,经高效液相色谱仪纯化后,制备成了命名为为F2ZGP96060801的大熊猫基因指纹探针,用同位素标记法标记F2ZGP96060801,LZF-I、朋5、33.6和(CAC)6/GTG)5等5种基因指纹探针,比较检测了大熊猫随机个体的被毛、大熊猫3雄配I雌所产1个双胞胎计6只个体的福尔马林固定的肝组织和粪便中的胃肠 相似文献
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The precise location of the SRY gene on the human Y chromosome has been revealed through studies of sex reversal cases involving deletion, cross-linking and mutations of the SRY gene. Its DNA sequence and mechanism of action are being understood. Similarity of SRY with Sry of mice and its interaction with other genes in male sex determination are discussed. 相似文献
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Fluorescent DNA probes (cCAT-F1 and cTAM-Fl) complementary to the 3′ end of ribosomal RNA (rRNA) internal transcribed spacer 1 sequences (ITS 1: positions 154–176) of toxic species of Alexandrium catenella (Whedon and Kofoid) Taylor and A. tamarense (Lebour) Taylor were applied to various cultures of the genus Alexandrium and several other phytoplankters using whole-cell fluorescent in situ hybridization. cCAT-F1 and cTAM-F1 reacted with targeted strains of A. catenella (catenella type) and A. tamarense (tamarense type), respectively, and did not react with isolates of A. affine (Inoue et Fukuyo) Balech, A. fraterculus (Balech) Balech, A. insuetum Balech, A. lusitanicum Balech, A. pseudogonyaulux (Biecheler)Horiguchi ex Yuki et Fukuyo comb. nov., nor isolates of Prorocentrum micans Ehrenberg, Amphidinium carterae Hulburt, Heterocapsa triquetra (Ehrenberg) Stein, Gymnodinium mikimotoi Miyake et Kominami ex Oda, Skeletonema costatum (Greville) Cleve, Heterosigma akashiwo (Hada) Hada, and Chattonella antiqua (Hada) Ono. DNase I and RNase A treatment showed that probes hybridized to ribosomal DNA, not rRNA. Probes were localized at the bottom of the U-shaped nucleus, a region that corresponds to the nucleolus. The probes are highly specific for particular strains of A. catenella and A. tamarense and are applicable for identifying these species collected from cultured and possibly natural populations. 相似文献
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为了研究X射线与X染色体的微校率之间的关系.本实验利用原位杂交技术同时检测了经X射线诱发人双核淋巴细胞的7号和X染色体的微核率。结果发现:经2.5Gy的X射线照射后.X和7号染色体的微核率男性分别为3.4%和7.1%;女性分别为6.6%和6.0%。X和7号染色体微核率的实验观察值与理论预期值之间在统计学上无显著性差异。实验结果提示:X射线并不特异性引起X染色体的微核率增高。 相似文献
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DNA指纹在近交系动物遗传污染检测中的应用 总被引:2,自引:0,他引:2
在用DNA指纹为建立葡萄胎动物模型选择动物的过程中,发现“三种”不同来源的BALB/C小鼠的DNA指纹出现了明显差别。与标准的保种品系B1和B2有12kb各缺铁了一条DNA片段,B1还缺失了6.6kb的片段,而在6kb处B1和B2都增加了一条DNA片段。 相似文献
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Christopher A. Scholin Roman Marin III Peter E. Miller Gregory J. Doucette Christine L. Powell Paul Haydock Judith Howard Jason Ray 《Journal of phycology》1999,35(6):1356-1367
Large-subunit ribosomal RNA-targeted probes for Pseudo-nitzschia australis Frenguelli, P. multiseries (Hasle) Hasle, P. pseudodelicatissima (Hasle) Hasle, and P. pungens (Grunow) Hasle were applied to cultured and natural samples using whole-cell and sandwich hybridization. Testing of the latter method is emphasized here, and technique refinements that took place during 1996–1997 are documented. Application of the sandwich hybridization test showed that the signal intensity obtained for a given number of target cells remained constant as batch cultures of these organisms progressed from active through stationary growth phases. This suggests that cellular rRNA content for each target species remained relatively stable despite changes in growth state. Application of whole-cell and sandwich hybridization assays to natural samples showed that both methods could be used to detect wild P. australis, P. pseudodelicatissima, and to a lesser degree P. multiseries, but detection of P. pungens was prone to error. A receptor-binding assay for domoic acid (DA) enabled detection of this toxin activity associated with a particulate fraction of the plankton and provided a context in which to view results of the rRNA probe tests. In one case, the probe for P. australis cross-reacted with P. cf. delicatissima. The sample that contained the latter species also contained a low amount of DA activity. Under certain field conditions, results of whole-cell and sandwich hybridization tests disagreed. Detailed analysis of selected field samples illustrates how such situations arose. Collectively, the rRNA probe and toxin analyses suggest that manifestation of DA in the environment is possible in the absence of readily recognizable intact cells. 相似文献