全文获取类型
收费全文 | 9699篇 |
免费 | 549篇 |
国内免费 | 339篇 |
出版年
2023年 | 93篇 |
2022年 | 102篇 |
2021年 | 139篇 |
2020年 | 243篇 |
2019年 | 286篇 |
2018年 | 309篇 |
2017年 | 237篇 |
2016年 | 207篇 |
2015年 | 224篇 |
2014年 | 697篇 |
2013年 | 968篇 |
2012年 | 570篇 |
2011年 | 496篇 |
2010年 | 305篇 |
2009年 | 423篇 |
2008年 | 423篇 |
2007年 | 484篇 |
2006年 | 429篇 |
2005年 | 426篇 |
2004年 | 302篇 |
2003年 | 293篇 |
2002年 | 249篇 |
2001年 | 236篇 |
2000年 | 182篇 |
1999年 | 190篇 |
1998年 | 213篇 |
1997年 | 166篇 |
1996年 | 177篇 |
1995年 | 226篇 |
1994年 | 233篇 |
1993年 | 179篇 |
1992年 | 164篇 |
1991年 | 99篇 |
1990年 | 88篇 |
1989年 | 59篇 |
1988年 | 54篇 |
1987年 | 40篇 |
1986年 | 37篇 |
1985年 | 46篇 |
1984年 | 53篇 |
1983年 | 33篇 |
1982年 | 40篇 |
1981年 | 34篇 |
1980年 | 36篇 |
1979年 | 25篇 |
1978年 | 14篇 |
1977年 | 14篇 |
1976年 | 14篇 |
1975年 | 7篇 |
1973年 | 8篇 |
排序方式: 共有10000条查询结果,搜索用时 17 毫秒
91.
Alec Breen Alan F. Rope Denise Taylor John C. Loper P. R. Sferra 《Journal of industrial microbiology & biotechnology》1995,14(1):10-16
Summary The use of DNA amplification fingerprinting (DAF) as a tool for monitoring mixed microbial populations in bioreactors was evaluated. Short (8-mer or 10-mer) oligonucleotides were used to prime DNA extracts from various biological reactors during polymerase chain reaction (PCR) amplification. The reactors examined in this study included two sets of anaerobic stirred tank continuous flow bioreactors. One set of anaerobic reactors was operated under methanogenic conditions and one set was operated under sulfate-reducing conditions. The anaerobic reactor communities in the methanol-fed reactors showed extensive DAF homology. DAF was also applied to a fixed-film azo dye degrading reactor to examine the degree of uniformity of colonization of the substratum in representative regions of the reactor. This method is a quick and relatively inexpensive means of monitoring microbial community structure during biological processes. Since no cultivation of the sample is involved, the genetic profile of the community is not biased by outgrowth conditions. DAF profiles may be useful for comparisons of population changes over time or of bench-scale vs pilot-scale reactors but not adequate for assessing community diversity. 相似文献
92.
二核苷酸重复多态性的非同位素检测及其在基因诊断中的应用 总被引:9,自引:1,他引:8
本文报道了一种检测二核苷酸重复多态性的简便的非同位素法,利用重复序列两侧的特异引物进行PCR扩增,产生的等位片段在薄层变性聚丙烯酰胺凝胶电泳上分离,再用灵敏的银染法显色。该方法不需要标记PCR产物,简便、快速,分辨率可达1bp,并可用多对引物同时进行多重PCR分析。用此方法对DMD家系成员dystrophin基因的5'-脑型外显子止游区和3'-非翻译区的两个(CA)。位点进行了扩增片段长度多态性分 相似文献
93.
用抗单纯疱疹病毒(HSV)型共同性gC和gD羊克隆抗体(McAb),包被即Eppendorf管,捕捉HSV,同时加入3个引物:一个是HSV─1/HSV─2型共同性上游引物,另两个分别是HSV─1和HSV─2型特异性下游引物。借此建立了能直接分型检测HSV的抗原捕获聚合酶链式反应(AC─PCR)。HSV─1的扩增产物为477bp,HSV─2的为399bp两型病毒经AC─PCR扩增后产生分子量不同的DNA片段,致使AC─PCR能直接分型检测HSV。HSV─1和HSV─2扩增产物的克隆和序列分析表明,本方法特异性好。用本法检测Balb/c幼鼠中枢神经系统HSV感染的脑标本,进一步证实本方法不仅敏感、特异,而且分型准确。 相似文献
94.
95.
利用PCR方法对单纯疱疹病毒Ⅱ型糖蛋白D(HSV-2gD)基因进行了修饰,在其5'端删去约500bp的非编码区,仅保留ATG上游7个bp。将修饰后的HSV-2gD基因插入到带有痘苗病毒天坛株TK基因区段的痘苗表达质粒pJSA1175,置于痘苗病毒P7.5k早/晚期启动子控制下。将此重组质粒用脂质体Lipofectin方法转染已受野型TK ̄+痘苗病毒天坛株感染的TK ̄-143细胞,通过同源重组机制和标志基因LacZ产物的蓝斑显色作用,以及BudR试剂对TK表型的选择压力,筛选出整合有HSV-2gD基因的重组痘苗病毒。Southem杂交表明,HSV-2gD基因已正确地插入痘苗病毒TK基因区内;间接免疫荧光检测显示,HSV-2gD蛋白已得到有效表达,且主要分布于细胞膜。重组病毒免疫家兔可产生明显的抗HSV-2gD中和抗体。用重组病毒免疫小鼠,3周后可使94%(17/18)的小鼠对抗HSV-2的致死量攻击,表明重组病毒具有明显的免疫保护作用。 相似文献
96.
应用PCR技术扩增出HBVDNAC基因片段并与pAT153质粒重组,转化到E.coliRRI中,经体内扩增,提纯,用光生物素标记,制备了C基因的重组质粒探针。该探针检测灵敏度在Southern印迹中达1pg,在点印迹中为5pg。用此探针以Southern印迹方式配合PCR技术检测乙肝病人血清中的HBVDNA,在53例PCR产物电泳检测阴性的样品中,Southern杂交又检出18例阳性。 相似文献
97.
PCR法快速检测临床标本中结核杆菌DNA 总被引:2,自引:0,他引:2
应用聚合酶链反应(PCR)快速检测临床标本(脑脊液、胸水、腹水、血、痰液)中的结核杆菌DNA,特异性扩增片段123bp,为结核杆菌的特异性重复序列IS6110部分基因。PCR检测人型结核杆菌的敏感性达10fgDNA。临床标本的PCR检测阳性率(23.3%)明显高于抗酸染色涂片(2.9%)和细菌培养(5.7%)的阳性率(P〈0.05)。通过设立对照系统及对扩增产物酶切分析,表明该法无假阴性结果(特异 相似文献
98.
伤寒Vi多糖菌苗接种反应观察 总被引:3,自引:1,他引:2
本文报告了对我国首次成功的伤寒Vi多糖菌苗进行人体接种反应观察结果,接种对象为20至54岁无伤寒病史,近年无伤寒菌苗接种史的健康人,共60名,以完全随机的方法分为两组,实验组注射30μgVi多糖菌苗,对照组注射Vi多糖菌苗的稀释液。其结果30名Vi多糖菌苗接种者注射后体温无中重反应发生,局部红肿仅有1例中反应。注射后对血压、心律没有影响。红细胞计数,白细胞计数均在正常范围,与接种前相比,无显著差异 相似文献
99.
Human follicular fluid (hFF), which has been treated with either unspecific proteases or dextran-coated charcoal (DCC) to remove proteins and/or steroids, cannot successfully induce the acrosome reaction (AR). After the removal of steroids, AR-inducing activity can be restored to hFF by supplementation with exogenous progesterone, but only in the presence of intact protein. Gel filtration experiments with 3H-progesterone-labelled hFF showed elution of the radioactive signal in the high molecular weight range, corresponding to bound progesterone. AR-inducing activity was seen in exactly the same fraction. Based on these results, the acrosome reaction-inducing substance (ARIS) appears to be a complex of progesterone and a progesterone-binding protein, which was shown to be identical with the plasma protein corticosteroid-binding globulin (CBG) by immunological techniques. AR induction was only observed in the presence of both CBG and progesterone, suggesting a combined effect of the two components. © 1995 wiley-Liss, Inc. 相似文献
100.
The competition method in which the Fenton reaction is employed as an OH radical generator and deoxyribose as a detecting molecule, has been used to determine the rate constants for reactions of the OH radical with its scavengers. Nonlinear competition plots were obtained for those scavengers which reacted with the Fenton reagents (Fe2+ or H2O2). Ascorbic acid is believed to overcome this problem. We have investigated the kinetics of deoxyribose degradation by -OH radicals generated by the Fenton reaction in the presence of ascorbic acid, and observed that the inclusion of ascorbic acid in the Fenton system greatly increased the rate of OH radical generation. As a result, the interaction between some scavengers and the Fenton reagents became negligeable and linear competition plots of A7A vs scavenger concentrations were obtained. The effects of experimental conditions such as, the concentrations of ascorbic acid, deoxyribose, H2O2 and Fe2+-EDTA, the EDTA/Fe2+ ratio as well as the incubation time, on the deoxyribose degradation and the determination of the rate constant for mercaptoethanol chosen as a reference compound were studied. The small standard error, (6.76± 0.21) ±' 109M-1s-1 observed for the rate constant values for mercaptoethanol determined under 13 different experimental conditions, indicates the latter did not influence the rate constant determination. This is in fact assured by introducing a term, kx, into the kinetic equation. This term represents the rate of-OH reactions with other reagents such as ascorbic acid, Fe2+-EDTA, H2O2 etc. The agreement of the rate constants obtained in this work with that determined by pulse radiolysis techniques for cysteine, thiourea and many other scavengers, suggests that this simple competition method is applicable to a wide range of compounds, including those which react with the Fenton reagents and those whose solubility in water is low. 相似文献