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犊牛腹泻粪样中牛呼肠孤病毒的分离鉴定 总被引:1,自引:0,他引:1
通过在培养液中添加胰蛋白酶的方法,用MA104细胞从犊牛腹泻粪样中分离并鉴定了1株能稳定产生细胞病变(CPE)的牛呼肠孤病毒,命名为B-19株.RNA聚丙烯酰胺凝胶(PAGE)电泳可见呼肠孤病毒典型的10条核酸节段,呈3:3:4排列.电镜检测结果显示,病毒粒子呈典型呼肠孤病毒粒子形态,直径约70 nm;病毒L1基因保守区段RT-PCR检测及序列分析表明,扩增出的440 bp 目的片段符合预期大小,而且其核苷酸序列与GenBank参考毒株L1基因序列具有较高同源性;S1基因序列分析表明,B-19株属于呼肠孤病毒血清1型(ST1). 相似文献
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<正>应用电泳方法研究家蚕血液蛋白始于五十年代,通过纸上电泳或用提塞留斯氏电泳仪(Tiselius apparatus)不连续电泳法等进行电泳,认为家蚕血蛋白由五种成分组成,近年来用聚丙烯酰胺凝胶盘状电泳法,最多能分离出17—18条带,其中有9条主带(何家禄等).家蚕血蛋白成分的电泳图谱,因电泳方法而异,聚丙烯酰胺凝胶盘状电泳法,分离效果高,图谱清晰. 相似文献
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为了研究不同生理状态下的卵巢卵母细胞基因的表达情况,采集乳牛的卵巢,分离了卵母细胞,以单个卵母细胞的mRNA作为模板,用设计的随机引物和锚定引物,采用一步法RT-PCR扩增了卵母细胞基因。经聚丙烯酰胺凝胶电泳检测,发现有功能黄体和无黄体卵巢卵母细胞基因的电泳条带之间无明显差异,对一条明显差异条带进行回收、纯化,将纯化的PCR产物连接到T载体,阳性克隆经鉴定后,进行测序和同源性比较。结果显示,与体外培养的牛早期胚胎的一条序列具有很高的同源性。 相似文献
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蒺藜苜蓿SSR反应体系优化及在一年生苜蓿种质鉴定中的应用 总被引:4,自引:2,他引:2
利用正交设计和完全随机试验优化蒺藜苜蓿(Medicago truncatula Gaertn)SSR标记的PCR反应体系。结果表明,适合于一年生苜蓿(Medicago L.)遗传分析的SSR技术体系为:10μL反应体系中TaqDNA聚合酶为1U,dNTP浓度为0.20mmol/L,Mg2+浓度为2.0mmol/L,引物浓度为1.5umol/L,模板DNA用量为20ng/uL;利用该反应体系将2个蒺藜苜蓿亲本和杂交种有效鉴别;利用SSR标记对19个一年生苜蓿种质进行SSR-PCR扩增,用8%的非变性聚丙烯酰胺凝胶电泳检测,不同种质间DNA谱带多态性丰富,通过UPGMA方法聚类分析可以明确区分19个一年生苜蓿种质。 相似文献
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PEG胁迫对尖叶胡枝子幼苗SOD和POD同工酶的影响 总被引:8,自引:3,他引:5
利用聚丙烯酰胺凝胶电泳技术研究不同浓度PEG处理尖叶胡枝子幼苗叶片SOD和POD同工酶及酶活性的变化。结果表明,不同浓度PEG处理,SOD和POD同工酶谱带数目没有变化,无新酶带的出现或酶带的减少,分别为SOD1-4和POD1-3。由迁移率可以看出,尖叶胡枝子幼苗叶片在不同浓度PEG处理下,共有10种SOD同工酶基因表达和6种POD同工酶基因表达。SOD和POD酶活性在低浓度(5~10%)PEG处理下增加,高浓度(15%)的PEG处理下降低。 相似文献
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苇状羊茅与多年生黑麦草内生真菌菌丝基因组DNA提取的优化及PCR初步检测 总被引:2,自引:1,他引:1
感染内生真菌的禾草在牧草和草坪业上具有重要的生态和经济意义,家畜采食感染Neotyphodiumcoenophialum和N.lolii的苇状羊茅Festuca arundinacea和多年生黑麦草Lolium perenne会发生中毒。研究收集天津口岸1998年以来进境的部分苇状羊茅和多年生黑麦草种子,对经镜检确认带有内生真菌的种子进行分离培养,对疑似菌株的菌丝用改进的Moller等方法进行基因组DNA抽提,测定浓度及纯度,对照原方法,DNA的纯度有较大提高,浓度略有上升。Tubulin2基因的引物IS1~IS3扩增结果显示为单一的条带,结合形态学和序列比对,分离培养得到的菌株可以基本确定为N.coenophialum和N.lolii。根据Gen-bank中N.coenophialum和N.lolii的NC25基因序列设计出引物F1~R1,扩增得到能区分开N.coenophialum和N.lolii的单一条带(相差160 bp),建立了N.coenophialum和N.lolii的PCR检测方法,结果准确可靠。 相似文献
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本研究通过核酸探针与免疫层析相结合的方法,建立了一种简单、敏感、特异的检测口蹄疫病毒的方法。试验利用RT-PCR方法获得口蹄疫病毒3D核苷酸片段,在引物中设计了灵敏度高、特异性好的核酸探针--生物素和地高辛,使扩增产物结合探针。利用胶体金的放大原理将链霉亲和素与金颗粒形成胶体金混合物,从而与RT-PCR产物中的生物素探针结合。硝酸纤维素膜上端标记生物素化山羊抗兔IgG作为指控条带,下端标记抗地高辛抗体以捕获RT-PCR产物中的地高辛探针。组装金标试纸条,检测RT-PCR产物,结果表明该核酸试纸条可以检测到 0.3×10-3~3×10-3 μg/μL,敏感性高于琼脂糖凝胶电泳,两种方法的符合率高,核酸试纸条检测方法是一种敏感性高、成本低且费时短的新型检测方法。该方法为口蹄疫病毒检测提供了一种新的思路。 相似文献
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In this article, through the combination of nucleic acid probes and immune chromatography, a simple, sensitive and specific detection system——nucleic acid lateral flow immunoassay (NALFIA) for amplifing foot-and-mouth disease virus (FMDV) 3D RT-PCR products was established.An ultrasensitive nucleic acid biosensor (NAB) based on streptavidin-labeled gold nanoparticles dual labels and lateral flow strip biosensor (LFSB) were used in this system.The biotinylated goat anti-rabbit IgG was marked to the NC membrane as the alleged strip and the anti-digoxin antibody was labeled to the NC membrane to capture the digoxin probe.After assemblying gold-labeled strip and detecting RT-PCR products, the detection limit of NALFIA was 0.3×10-3 to 3×10-3 μg/μL.The NALFIA was compared with agar gel electrophoresis analysis, the results showed that the sensitivity of NALFIA was higher than agar gel electrophoresis.There was an excellent agreement between the two methods.NALFIA was a method with high sensitive, low cost and short time.In conclusion, this method provided a good alternative to detect FMDV. 相似文献
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T Hohdatsu M Yamada M Okada M Fukasawa K Watanabe T Ogasawara M Takagi C Aizawa M Hayami H Koyama 《Veterinary microbiology》1992,30(2-3):113-123
Feline immunodeficiency virus (FIV) proviral DNA was detected by the polymerase chain reaction method (PCR). PCR products were detected by gel electrophoresis and ethidium bromide staining. The P-10, P-15 and P-24 regions of the gag gene of FIV were chosen as the target sequences for amplification, and three primer pairs were prepared. The PCR products subjected to amplification with each primer pair were found to possess sites of digestion by a restriction enzyme, as hypothesized. They did not react with feline leukemia virus (FeLV)-infected or feline syncytium-forming virus (FeSFV)-infected cell-derived DNA, and specifically amplified FIV-infected cell-derived DNA. FIV proviral DNA was detected by the PCR method with either primer pair (one-step amplification: single PCR) in DNA derived from peripheral blood lymphocytes (PBL) from 7 of 12 FIV antibody-positive cats. When PCR products in each of the 12 cats were subjected to a second amplification using the same primer pair (two-step amplification: double PCR), FIV proviral DNA was detected in all of the cats. When PBL samples collected from three cats that were negative and three that were positive in the single PCR were cultured for a few weeks in the presence of interleukin 2, FIV proviral DNA was detected in all six cats by the single PCR method. The results suggest that either the use of cultured PBL as the sample or the performance of the double PCR method enables simple and specific detection of FIV proviral DNA in PBL. 相似文献
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Construction of an internal standard used in RT nested PCR for Borna Disease Virus RNA detection in biological samples 总被引:4,自引:0,他引:4
The highly neurotropic Borna Disease Virus (BDV), which belongs to the Mononegavirales order--Bornaviridae family--is generally detected using the RT-nested-PCR. If false positive results (often caused by laboratory contaminations) can be avoided, some false negative results which are mostly due to inhibitory effects of some reaction components and/or to sample preparation errors, can occur. Thus, in order to control the RT-PCR sample, an RNA internal standard molecule named "mimic" was constructed with the same primer recognition sites as the viral nucleic acids, flanking a heterologous DNA fragment of distinct molecular weight. Because of their different sizes, the mimic and viral PCR products can be easily discriminated by agarose gel electrophoresis. The co-amplification of both BDV and mimic RNA was performed on infected cells and on biological tissues such as the brain and blood, commonly known to contain PCR inhibitor components. After mimic sensitivity studies were achieved (2.5 fg of "p40 RNA mimic" and 0.25 fg of "p24 RNA mimic"), the competitive amplification reaction between both BDV and mimic RNA was performed on these tissues. The results confirmed that nervous tissue has an inhibitory effect on RT-PCR, which supports the necessity of BDV detection by a higher sensitive method such as RT nested PCR. Moreover, these results confirmed the interest of an internal standard for BDV RNA detection in biological samples. 相似文献
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用RAPD标记分析部分柞蚕二化性品种资源的遗传多样性 总被引:3,自引:2,他引:1
采用RAPD标记技术,对36份柞蚕(Antheraea pernyi)二化性品种资源的遗传多样性进行分析。PCR扩增及琼脂糖凝胶电泳结果表明:利用筛选出的19个随机引物在供试材料中共检测到123个信息位点,其中多态性位点114个,多态性比率92.68%;每一个引物可检测出3~9个位点,其多态性比率不尽相同,平均扩增出6.47条带;各品种具有不同数量的特异性带型。供试品种资源的Nei氏遗传多样性指数(h)为0.324 3±0.151 5,Shannon信息指数(I)为0.487 1±0.197 5,表明长期同地保存的二化性柞蚕品种资源同样具有丰富的遗传多样性;供试品种的Nei氏遗传一致性范围在0.495 9~0.837 4之间,达0.6以上的占96.59%,表明多数品种资源的相似程度较高。36份品种资源分别以辽宁省蚕业科学研究所育成种为主和以国内外引进种为主聚为两大类群。 相似文献
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分别以NDV F48E9和La Sota株感染鸡胚成纤维细胞,于感染后8 h提取NDV感染CEF细胞总RNA,通过mRNA差异显示技术筛选病毒感染诱导表达的特异性基因.主要方法是先以锚定引物经反转录后,然后以9~10 bp随机引物为上游引物,以锚定引物Oligod(T)18为下游引物,进行PCR扩增,PCR产物采用8%的尿素变性PAGE胶电泳进行分离鉴定.对于特征性差异带进行第二次PCR扩增,克隆后测定其核苷酸序列.结果我们发现数个差异条带,选取差异带在500 bp以内的条带,经测序后显示经NDV F48E9感染后,有一条差异条带特异性表达的基因所编码的蛋白与Receptor(TNFRSF)-interacting serine-threonine kinase 2有47 %同源,是一个新基因,其蛋白功能不明确,有可能和细胞的凋亡有关. 相似文献